Designer ligands of tgf-beta superfamily

ABSTRACT

The present disclosure relates to chimeric polypeptide having TGF-beta activity, nucleic acids encoding the polypeptides, and host cells for producing the polypeptides.

CROSS REFERENCE TO RELATED APPLICATIONS

The application claims priority under 35 U.S.C. §119 to U.S. ProvisionalApplication Ser. No. 61/155,066, filed, Feb. 24, 2009, the disclosure ofwhich is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The invention was funded, in part, by a grant from the NationalInstitutes of Health grant number HD013527. The government has certainright in this invention.

TECHNICAL FIELD

The present disclosure relates to biomolecular engineering and design,and engineered proteins and nucleic acids.

BACKGROUND

Activins and Bone Morphogenetic Proteins (BMPs) are members of the muchlarger Transforming Growth Factor-beta (TGF-β) superfamily. Due to theirpervasiveness in numerous developmental and cellular processes, TGF-βligands have been the focus of great interest. For TGF-β ligands to besuccessfully used as therapeutic tools, several hurdles need to beovercome. The ability to specifically modify and alter the properties ofTGF-β ligands, as well as generate those ligands in significantquantities is required.

SUMMARY

The disclosure provides non-naturally occurring chimeric polypeptideshaving an activity provided by a TGF-beta family of proteins. Thechimeric polypeptides of the disclosure comprises two or more segmentsor fragments from parental TGF-beta proteins operably linked such thatthe resulting polypeptide is capable of modulating a pathway associatedwith a TGF-beta family of proteins. In one embodiment, the pathway is aSMAD or DAXX pathway.

In one embodiment, the disclosure provides designer TGF-beta ligandsthat can be synthesized by selecting and conjoining different sequencesegments of TGF-beta superfamily ligands to construct new ligands(designer ligands). These novel ligands possess entirely new proteinsequence library that differs from naturally existing TGF-betasuperfamily ligands. This approach originates primarily from therecognition of the structural commonality among natural TGF-betasuperfamily ligands. All ˜40 TGF-beta superfamily ligands share the sameoverall architecture with generic characteristics for each region of theprotein. The framework of TGF-beta ligands can be divided into(generally) six subdomains (also called sequence segments; marked in sixdifferent colors in FIG. 19) that all superfamily members share.

In one embodiment, the disclosure also provides a recombinantpolypeptide comprising: at least two peptide segments, a first segmentof the polypeptide comprising a sequence having at least 80% identity toa first TGF-beta family protein and a second segment comprising asequence having at least 80% identity to a second TGF-beta familyprotein, wherein the segments are operably linked and have activity ofat least one of the first or second parental TGF-beta family protein. Inone embodiment, the polypeptide comprises an N-terminal segment fromBMP-2. In another embodiment, the at least two peptide segments comprise6 peptide segments operably linked N-terminus to C-terminus. In yetanother embodiment, each of the first and second TGF-beta familyproteins have structural similarity and each segment corresponds to astructural motif. In yet a further embodiment, the first TGF-beta familyprotein is BMP-2 and the second TGF-beta family protein is activin. Inone embodiment, the segments of the BMP-2 protein comprise segment 1:amino acid residue from about 1 to about x₁ of SEQ ID NO:2 (“1b”);segment 2 is from about amino acid residue x₁ to about x₂ of SEQ ID NO:2(“2b”); segment 3 is from about amino acid residue x₂ to about x₃ of SEQID NO:2 (“3b”); segment 4 is from about amino acid residue x₃ to aboutx₄ of SEQ ID NO:2 (“4b”); segment 5 is from about amino acid residue x₄to about x₅ of SEQ ID NO:2 (“5b”); and segment 6 is from about aminoacid residue x₅ to about x₆ of SEQ ID NO:2 (“6b”); and wherein: x₁ isresidue 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 of SEQ ID NO:2; x₂is residue 45, 46, 47, or 48 of SEQ ID NO:2; x₃ is residue 65, 66, 67,or 68 of SEQ ID NO:2; x₄ is residue 76, 77, 78, 79, 80, 81 or 82 of SEQID NO:2; x₅ is residue 88, 89, 90, 91, 92, 93, or 94 of SEQ ID NO:2; andx₆ is residue 112, 113, or 114 or SEQ ID NO:2, corresponding to theC-terminus of BMP-2; and the segments of the activin protein comprisesegment 1, amino acid residue from about 1 to about x₁ of SEQ ID NO:5(“1a”); segment 2 is from about amino acid residue x₁ to about x₂ of SEQID NO:5 (“2a”); segment 3 is from about amino acid residue x₂ to aboutx₃ of SEQ ID NO:5 (“3a”); segment 4 is from about amino acid residue x₃to about x₄ of SEQ ID NO:5 (“4a”); segment 5 is from about amino acidresidue x₄ to about x₅ of SEQ ID NO:5 (“5a”); and segment 6 is fromabout amino acid residue x₅ to about x₆ of SEQ ID NO:5 (“6a”); andwherein: x₁ is residue 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 ofSEQ ID NO:5; x₂ is residue 42, 43, 44, or 45 of SEQ ID NO:5; x₃ isresidue 61, 62, 63, or 64 of SEQ ID NO:5; x₄ is residue 78, 79, 80, 81,82, 83 or 84 of SEQ ID NO:5; x₅ is residue 90, 91, 92, 93, 94, 95 or 96of SEQ ID NO:5; and x₆ is residue 114, 115, or 116 or SEQ ID NO:5; andwherein the polypeptide has an order of segment 1-segment 2-segment3-segment 4-segment 5-segment 6. In a further embodiment, thepolypeptide comprises a sequence of segments selected from the groupconsisting of 1b2b3b4b5b6a; 1b2b3b4b5a6a; 1b2b3b4b5a6b; 1b2b3a4a5a6a;1b2b3a4a5b6a; 1b2a3a4a5a6a; 1b2a3a4a5a6a L66V/V67I; 1b(1a_II)2a3a4a5a6a;1b2a3a4a5a6b; 1b2a3a4a5b6b; 1b2a3a4a5b6a; 1b2a3b4b5b6a; 1b2a3b4b5a6a;and 1b2a3b4b5a6b.

The disclosure also provides a recombinant polypeptide comprising atleast 80%, 90%, 95%, 98% or 99% identity to a sequence as set forth inSEQ ID NO:7, 9, 11, 13, 15, 17, 19, 1, 23, 25, 2, 29, 31, 33, 35, 37, 39or 41 and wherein the polypeptide modulates the SMAD or DAXX pathway.

The disclosure also provides a chimeric TGF-beta family polypeptidecomprising a segment of a first TGF-beta family protein operably linkedto a segment of a second different TGF-beta family protein to provide achimeric polypeptide having SMAD or DAXX modulating activity.

The disclosure also provides a polynucleotide encoding a polypeptide ofthe disclosure. In one embodiment, the polynucleotide has at least 80%,90%, 95%, 98%, 99% or more identity to a sequence selected from thegroup consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 24, 26, 28, and 40. Vectors comprising such polynucleotidesare also provided along with recombinant cells.

In an alternate embodiment, the disclosure provides novel ligands formembers of the TGF-beta superfamily, wherein the ligand is a chimericprotein with at least one of six subdomains from a foreign or differentmember of the TGF-beta superfamily.

The disclosure comprises a chimeric polypeptide comprising a firstdomain from a first TGF-beta family member, a crossover point at J1(see, e.g., FIG. 18), a second domain from the same or second TGF-betafamily member, a crossover point at J2 (see, e.g., FIG. 18), a thirddomain from the same or third TGF-beta family member, a crossover pointat J3 (see, e.g., FIG. 18), a fourth domain from the same or fourthTGF-beta family member, a crossover point at J4 (see e.g., FIG. 18), afifth domain from the same or fifth TGF-beta family member, a crossoverpoint at J5 (see, e.g., FIG. 18), and a second domain from the same orsixth TGF-beta family member. In one embodiment, the chimera is derivedfrom 2, 3, 4, 5, or 6 different TGF-beta family members. In yet anotherembodiment a crossover at J3 is optional.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-B shows BMP2/BMP6 sample characterization. Panel A: BMP2/BMP6sample on SDS-PAGE. The BMP2/BMP6 sample non-reduced in lane one,molecular weight marker in lane two, and the BMP2/BMP6 sample reduced inlane three. Panel B: SELDI-TOF-MS overlaid results from separate samplesof BMP2, BMP6, and BMP2/BMP6 ligands.

FIG. 2 shows C₂Cl₂ whole cell Smad1-dependent reporter assay with wildtype ligands. The solid black bars represent error.

FIG. 3 shows a visualization of chick limb bud mesenchyme cell micromassculture chrondogenesis assays after 5 days. The top panel shows theculture with no factor and the extracellular antagonist Noggin. The barsin the micrographs represent 1 mm. The second through fourth panels showthe culture with ligands BMP2, BMP6, and BMP2/BMP6 respectively.

FIG. 4 shows quantification of chick limb bud mesenchyme cell micromassculture chrondogenesis assays after three days. The addition of thespecified growth factor at different concentrations is indicated.

FIG. 5 shows Mutant BMP2/BMP6 heterodimers displaying activation ofSmad1 reporter gene. Sample a contains no factor (background) and Sampleb contains a BMP2 homodimer with no active receptor sites. Allquantities are normalized to Sample c which is the fully activeBMP2/BMP6 heterodimer (100% activation of reporter gene).

FIG. 6 shows a native-PAGE displaying the quantity of type II receptorECD remaining after being saturated into a ligand-receptor complex withthe specified ligands.

FIG. 7 depicts a structure/sequence alignment for chimera designstrategy.

FIG. 8 provides a graph showing cell viability in the presence ofxerogel material (lowest concentration, green, 0.3 ug/ul; mediumconcentration, red, 3 ug/ul; high concentration, blue, 30 ug/ul).

FIG. 9 shows H9 hES cells cultured in mCIVA using differentconcentrations of AB2-008 in the absence or presence of human FGF2.

FIG. 10 shows mineralization shown by Von Kossa staining with (A) noligand added, (B) recombinant BMP2 (30 ng/ml), (B) AB2-004 (30 ng/ml),(C) AB2-011 (30 ng/ml) and (D) AB2-015 (30 ng/ml).

FIG. 11 shows the signaling activities of AB2-008, AB2-009, and AB2-010.(A) AB2-008 versus activin-βA (B) AB2-009 versus activin-βA (C) AB2-010versus activin-βA (D) Relative signaling strength in comparison toactivin-βA.

FIG. 12 shows phosphorylation by AB2-008, AB2-009, and AB2-010 incomparison to Activin-βA and BMP2. Activin-βA, AB2-008, and AB2-009 showcomparable levels of phosphorylation of SMAD2, whereas BMP2 showsphosphorylation of SMAD1 specifically.

FIG. 13 depicts FSH release by Activin-βA, AB2-008, AB2-009, andAB2-010. (A) Dose dependent FSH stimulation without Inhibin, and (B)decreased release with Inhibin.

FIG. 14 shows co-receptor binding by Activin-βA and AB2-008.Smad-2-Luciferase activity in HEK cells in the presence of and absenceCripto with (A) activin-βA, and with (B) AB2-008.

FIG. 15 shows signaling activity of AB2-011, AB2-012, and AB2-015 bySmad-1 pathway. (A) AB2-004 v.s. BMP2, (B) AB2-011 v.s. BMP2, (C)AB2-012 v.s. BMP2, and (D) AB2-015 v.s. BMP2, all in the concentrationrange of 3-30 ng/ml in culture media using Smad-1 Luciferase assay withC2C12 cells.

FIG. 16 shows Noggin sensitivity of BMP2, AB2-004, AB2-011, AB2-012, andAB2-015. Smad-1 luciferase signaling activity is measured with (lightgray) and without (dark gray) Noggin.

FIG. 17 is a schematic description of the RASCH method.

FIG. 18 provides an alignment of the sequences of several members of theTGF-beta superfamily, with the relative segments defined for eachmember.

FIG. 19 shows the six subdomains (fragments) on a single subunit of theTGF-beta superfamily ligand's scaffold.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a domain” includesa plurality of such domains and reference to “the protein” includesreference to one or more proteins, and so forth.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

Although methods and materials similar or equivalent to those describedherein can be used in the practice of the disclosed methods andcompositions, the exemplary methods and materials are described herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Thus, as used throughout theinstant application, the following terms shall have the followingmeanings.

“Amino acid” is a molecule having the structure wherein a central carbonatom (the alpha-carbon atom) is linked to a hydrogen atom, a carboxylicacid group (the carbon atom of which is referred to herein as a“carboxyl carbon atom”), an amino group (the nitrogen atom of which isreferred to herein as an “amino nitrogen atom”), and a side chain group,R. When incorporated into a peptide, polypeptide, or protein, an aminoacid loses one or more atoms of its amino acid carboxylic groups in thedehydration reaction that links one amino acid to another. As a result,when incorporated into a protein, an amino acid is referred to as an“amino acid residue.”

“Protein” or “polypeptide” refers to any polymer of two or moreindividual amino acids (whether or not naturally occurring) linked via apeptide bond, and occurs when the carboxyl carbon atom of the carboxylicacid group bonded to the alpha-carbon of one amino acid (or amino acidresidue) becomes covalently bound to the amino nitrogen atom of aminogroup bonded to the -carbon of an adjacent amino acid. The term“protein” is understood to include the terms “polypeptide” and “peptide”(which, at times may be used interchangeably herein) within its meaning.In addition, proteins comprising multiple polypeptide subunits (e.g.,DNA polymerase III, RNA polymerase II) or other components (for example,an RNA molecule, as occurs in telomerase) will also be understood to beincluded within the meaning of “protein” as used herein. Similarly,fragments of proteins and polypeptides are also within the scope of thedisclosure and may be referred to herein as “proteins.” In one aspect ofthe disclosure, a polypeptide comprises a chimera of two or moreparental peptide segments.

As used herein, TGF-beta superfamily member refers to a TGF-betasuperfamily (including bone morphogenic factors) gene or protein of anyspecies, particularly a mammalian species, including but not limited tobovine, ovine, porcine, murine, equine, and human. “TGF-beta superfamilypolypeptide” refers to the amino acid sequences of purified TGF-betasuperfamily protein obtained from any species, particularly a mammalianspecies, including bovine, ovine, porcine, murine, equine, and human andfrom any source, whether natural, synthetic, semi-synthetic, orrecombinant.

“Peptide segment” refers to a portion or fragment of a largerpolypeptide or protein. A peptide segment need not on its own havefunctional activity, although in some instances, a peptide segment maycorrespond to a domain of a polypeptide wherein the domain has its ownbiological activity. A stability-associated peptide segment is a peptidesegment found in a polypeptide that promotes stability, function, orfolding compared to a related polypeptide lacking the peptide segment.

A particular amino acid sequence of a given protein (i.e., thepolypeptide's “primary structure,” when written from the amino-terminusto carboxy-terminus) is determined by the nucleotide sequence of thecoding portion of a mRNA, which is in turn specified by geneticinformation, typically genomic DNA (including organelle DNA, e.g.,mitochondrial or chloroplast DNA). Thus, determining the sequence of agene assists in predicting the primary sequence of a correspondingpolypeptide and more particular the role or activity of the polypeptideor proteins encoded by that gene or polynucleotide sequence.

“Fused,” “operably linked,” and “operably associated” are usedinterchangeably herein to broadly refer to a chemical or physicalcoupling of two otherwise distinct domains, wherein each domain hasindependent biological function. As such, the present disclosureprovides TGF-beta (e.g., BMP or activins) domains that are fused to oneanother such that they function as a polypeptide having a TGF-betafamily activity or an improvement or change in ligand specificity of aTGF-beta family of polypeptides. In one embodiment, a chimericpolypeptide comprising a plurality of domains from two parental TGF-betafamily polypeptides are linked such that they are part of the samecoding sequence, each domain encoded by a polynucleotide from a parentalTGF-beta family polypeptide, wherein the polynucleotides are in framesuch that the polynucleotide when transcribed encodes a single mRNA thatwhen translated comprises a plurality of domains as a singlepolypeptide. Typically, the coding domains will be linked “in-frame”either directly of separated by a peptide linker and encoded by a singlepolynucleotide. Various coding sequences for peptide linkers and peptideare known in the art.

“Polynucleotide” or “nucleic acid” refers to a polymeric form ofnucleotides. In some instances a polynucleotide comprises a sequencethat is not immediately contiguous with either of the coding sequenceswith which it is immediately contiguous (one on the 5′ end and one onthe 3′ end) in the naturally occurring genome of the organism from whichit is derived. The term therefore includes, for example, a recombinantDNA which is incorporated into a vector; into an autonomouslyreplicating plasmid or virus; or into the genomic DNA of a prokaryote oreukaryote, or which exists as a separate molecule (e.g., a cDNA)independent of other sequences. The nucleotides of the disclosure can beribonucleotides, deoxyribonucleotides, or modified forms of eithernucleotide. A polynucleotides as used herein refers to, among others,single- and double-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Theterm polynucleotide encompasses genomic DNA or RNA (depending upon theorganism, i.e., RNA genome of viruses), as well as mRNA encoded by thegenomic DNA, and cDNA.

“Nucleic acid segment,” “oligonucleotide segment” or “polynucleotidesegment” refers to a portion of a larger polynucleotide molecule. Thepolynucleotide segment need not correspond to an encoded functionaldomain of a protein; however, in some instances the segment will encodea functional domain of a protein. A polynucleotide segment can be about6 nucleotides or more in length (e.g., 6-20, 20-50, 50-100, 100-200,200-300, 300-400 or more nucleotides in length).

“Chimera” or “chimeric protein” or “chimeric polypeptide” refers to acombination of at least two segments of at least two different parentproteins. As appreciated by one of skill in the art, the segments neednot actually come from each of the parents, as it is the particularsequence that is relevant, and not the physical nucleic acidsthemselves. For example, a chimeric BMP will have at least two segmentsfrom two different parent BMPs; or BMP and other member of the TGF-betasuperfamily, or alternatively, an unrelated protein. A chimeric proteinmay also be an “interspecies,” “intergenic,” etc. fusion of proteinstructures (the same or different member protein) expressed by differentkinds of organisms. In a one embodiment, two segments are connected soas to result in a new chimeric protein. In other words, a protein willnot be a chimera if it has the identical sequence of either one of thefull-length parents. A chimeric protein can comprise more than twosegments from two different parent proteins. For example, there may be2, 3, 4, 5, 6 or 10-20, or more parents from which the domains may bederived in generating a final chimera or library of chimeras. Thesegment of each parent protein can be very short or very long, thesegments can range in length of contiguous amino acids from 1 to aboutthe full length of the protein. In one embodiment, the minimum length is5 amino acids. Generally, the segment or subdomain, is one of sixsubdomains, alternatively five subdomains (see FIGS. 18 and 19). The sixsegments of a TGF-beta superfamily member are identified based on thestructural architecture of the member protein and/or the primary aminoacid sequence as aligned against other homologous member proteins. Asidentified, the member protein is generally divided into 6 distinctsections (although, alternatively, 5 distinct sections) based onsegments derived to minimize alterations, or alternatively viewed,maximize alterations, to the aligned native TGF-beta member sequenceduring chimera engineering. Generally, FIG. 18 shows the relativepositions of the distinct segments overlapping the aligned sequences ofeach of several TGF-beta superfamily members. The vertical line denotesa general position for cross-over between domains in generating thechimera. The amino acids that can overlap the two domains can be definedas being plus or minus about 5 amino acids (or alternatively, 8, 7, 6,5, 4, 3, 2 or 1 amino acids) in either direction of the vertical line.Also in FIG. 18 is shown a boxed set of amino acids that identifyadditional junctions that can be used to generate chimera. The J1-J5junctions are positions general conservation across the TGF-beta familyproteins that can be used to generate cross-over points.

Although relatively distinct, the segments may comprise a particularamino acid sequence or an original amino acid sequence that is amenableto substitution(s), insertion(s), additional amino acid(s) at either orboth termini of the original sequence, or other modifications. By“amenable”, it is meant that the structural integrity of each segment ismaintained as compared to the domain of the original sequence. Forexample, a segment described herein of a TGF-beta superfamily member mayshift by 10, 5, 3, 2, or 1, or preferably no more than 1 amino acid oneither or both termini of segment as identified.

In one embodiment, the disclosure provides a chimeric protein comprisinga fusion of at least one segment from a TGF-beta member with a secondsegment from a second TGF-beta member, wherein the first segment isforeign to the second TGF-beta member. Utilizing the five-six subdomains(segments) on a single subunit of the TGF-beta superfamily ligand as ascaffold framework, new (designer) sequences can be recombinantly linkedby mixing segments from different TGF-beta ligands in the same order asthey appear in nature. This assembly produces new sequences that arepartly similar to one of several different target sequences, butdistinctly different from any naturally occurring sequences.

In one embodiment, a single crossover point is defined for two parents.The crossover location defines where one parent's amino acid segmentwill stop and where the next parent's amino acid segment will start.Thus, a simple chimera would only have one crossover location where thesegment before that crossover location would belong to one parent andthe segment after that crossover location would belong to the secondparent. In one embodiment, the chimera has more than one crossoverlocation. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-30, or morecrossover locations. In a particular embodiment, the parental strandsare defined as having 4 or 5 crossover locations. How these crossoverlocations are named and defined are both discussed below. In anembodiment where there are two crossover locations and two parents,there will be a first contiguous segment from a first parent, followedby a second contiguous segment from a second parent, followed by a thirdcontiguous segment from the first parent. Contiguous is meant to denotethat there is nothing of significance interrupting the segments. Thesecontiguous segments are connected to form a contiguous amino acidsequence. For example, a BMP-2/activin chimera derived from a BMP-2wild-type parental strand and an activin wild-type parental strand withtwo crossovers would comprise a first segment from either BMP-2 oractivin, a second segment from the opposite parental strand compared tothe first segment operably linked to the first strand and comprising astructural motif downstream of the first strand and a third segmentstrand from the opposite parental strand compared to the second segmentand from the same parental strand as the first segment operably linkedto the second strand and comprising a structural motif downstream of thesecond strand all connected in one contiguous amino acid chain.

As appreciated by one of skill in the art, variants of chimeras exist aswell as the exact sequences. In other words conservative amino acidsubstitutions may be incorporated into the chimera (e.g., from about1-10 conservative amino acid substitutions). Thus, not 100% of eachsegment need be present in the final chimera if it is a variant chimera.The amount that may be altered, either through additional residues orremoval or alteration of residues will be defined as the term variant isdefined. Of course, as understood by one of skill in the art, the abovediscussion applies not only to amino acids but also nucleic acids whichencode for the amino acids.

“Conservative amino acid substitution” refers to the interchangeabilityof residues having similar side chains, and thus typically involvessubstitution of the amino acid in the polypeptide with amino acidswithin the same or similar defined class of amino acids. By way ofexample and not limitation, an amino acid with an aliphatic side chainmay be substituted with another aliphatic amino acid, e.g., alanine,valine, leucine, isoleucine, and methionine; an amino acid with hydroxylside chain is substituted with another amino acid with a hydroxyl sidechain, e.g., serine and threonine; an amino acids having aromatic sidechains is substituted with another amino acid having an aromatic sidechain, e.g., phenylalanine, tyrosine, tryptophan, and histidine; anamino acid with a basic side chain is substituted with another aminoacid with a basis side chain, e.g., lysine, arginine, and histidine; anamino acid with an acidic side chain is substituted with another aminoacid with an acidic side chain, e.g., aspartic acid or glutamic acid;and a hydrophobic or hydrophilic amino acid is replaced with anotherhydrophobic or hydrophilic amino acid, respectively.

“Non-conservative substitution” refers to substitution of an amino acidin the polypeptide with an amino acid with significantly differing sidechain properties. Non-conservative substitutions may use amino acidsbetween, rather than within, the defined groups and affects (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine) (b) the charge or hydrophobicity, or (c) the bulkof the side chain. By way of example and not limitation, an exemplarynon-conservative substitution can be an acidic amino acid substitutedwith a basic or aliphatic amino acid; an aromatic amino acid substitutedwith a small amino acid; and a hydrophilic amino acid substituted with ahydrophobic amino acid.

“Reference sequence” refers to a defined sequence used as a basis for asequence comparison. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence can be at least 20 nucleotideor amino acid residues in length, at least 25 residues in length, atleast 50 residues in length, or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptides aretypically performed by comparing sequences of the two polynucleotides orpolypeptides over a “comparison window” to identify and compare localregions of sequence similarity.

“Sequence identity” means that two amino acid sequences aresubstantially identical (i.e., on an amino acid-by-amino acid basis)over a window of comparison. The term “sequence similarity” refers tosimilar amino acids that share the same biophysical characteristics. Theterm “percentage of sequence identity” or “percentage of sequencesimilarity” is calculated by comparing two optimally aligned sequencesover the window of comparison, determining the number of positions atwhich the identical residues (or similar residues) occur in bothpolypeptide sequences to yield the number of matched positions, dividingthe number of matched positions by the total number of positions in thewindow of comparison (i.e., the window size), and multiplying the resultby 100 to yield the percentage of sequence identity (or percentage ofsequence similarity). With regard to polynucleotide sequences, the termssequence identity and sequence similarity have comparable meaning asdescribed for protein sequences, with the term “percentage of sequenceidentity” indicating that two polynucleotide sequences are identical (ona nucleotide-by-nucleotide basis) over a window of comparison. As such,a percentage of polynucleotide sequence identity (or percentage ofpolynucleotide sequence similarity, e.g., for silent substitutions orother substitutions, based upon the analysis algorithm) also can becalculated. Maximum correspondence can be determined by using one of thesequence algorithms described herein (or other algorithms available tothose of ordinary skill in the art) or by visual inspection.

As applied to polypeptides, the term substantial identity or substantialsimilarity means that two peptide sequences, when optimally aligned,such as by the programs BLAST, GAP or BESTFIT using default gap weightsor by visual inspection, share sequence identity or sequence similarity.Similarly, as applied in the context of two nucleic acids, the termsubstantial identity or substantial similarity means that the twonucleic acid sequences, when optimally aligned, such as by the programsBLAST, GAP or BESTFIT using default gap weights (described in detailbelow) or by visual inspection, share sequence identity or sequencesimilarity.

One example of an algorithm that is suitable for determining percentsequence identity or sequence similarity is the FASTA algorithm, whichis described in Pearson, W. R. & Lipman, D. J., (1988) Proc. Natl. Acad.Sci. USA 85:2444. See also, W. R. Pearson, (1996) Methods Enzymology266:227-258. Preferred parameters used in a FASTA alignment of DNAsequences to calculate percent identity or percent similarity areoptimized, BL50 Matrix 15: −5, k-tuple=2; joining penalty=40,optimization=28; gap penalty −12, gap length penalty=−2; and width=16.

Another example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity or percent sequence similarity. It also plots a treeor dendogram showing the clustering relationships used to create thealignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng & Doolittle, (1987) J. Mol. Evol. 35:351-360. The methodused is similar to the method described by Higgins & Sharp, CABIOS5:151-153, 1989. The program can align up to 300 sequences, each of amaximum length of 5,000 nucleotides or amino acids. The multiplealignment procedure begins with the pairwise alignment of the two mostsimilar sequences, producing a cluster of two aligned sequences. Thiscluster is then aligned to the next most related sequence or cluster ofaligned sequences. Two clusters of sequences are aligned by a simpleextension of the pairwise alignment of two individual sequences. Thefinal alignment is achieved by a series of progressive, pairwisealignments. The program is run by designating specific sequences andtheir amino acid or nucleotide coordinates for regions of sequencecomparison and by designating the program parameters. Using PILEUP, areference sequence is compared to other test sequences to determine thepercent sequence identity (or percent sequence similarity) relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps. PILEUP can be obtained fromthe GCG sequence analysis software package, e.g., version 7.0 (Devereauxet al., (1984) Nuc. Acids Res. 12:387-395).

Another example of an algorithm that is suitable for multiple DNA andamino acid sequence alignments is the CLUSTALW program (Thompson, J. D.et al., (1994) Nuc. Acids Res. 22:4673-4680). CLUSTALW performs multiplepairwise comparisons between groups of sequences and assembles them intoa multiple alignment based on sequence identity. Gap open and Gapextension penalties were 10 and 0.05 respectively. For amino acidalignments, the BLOSUM algorithm can be used as a protein weight matrix(Henikoff and Henikoff, (1992) Proc. Natl. Acad. Sci. USA89:10915-10919).

FIG. 18, for example, shows an alignment of a number of TGF-beta familymembers. One of skill in the art can readily determine from thealignment those amino acids that are conserved across the family as wellas those that are not conserved.

“Functional” refers to a polypeptide which possesses either the nativebiological activity of the naturally-produced proteins of its type, orany specific desired activity, for example as judged by its ability tobind to ligand or cognate molecules or induce a particular biologicalfunction (e.g., stimulate muscle growth, bone growth and the like).

The Transforming Growth Factor-beta (TGF-β) superfamily of proteins iscomprised of extracellular cytokines found in the vast majority of humancells. The TGF-β superfamily ligands, of which there are ˜40, can besubdivided into smaller families including TGF-β, Bone MorphogeneticProteins (BMPs), activin and inhibin, Growth and Differentiation Factors(GDFs), Nodal, Müllerian Inhibiting Substance (MIS), and Glial cellline-Derived Neurotrophic Factors (GDNFs). TGF-β superfamily members arefound in a diverse range of cell types and play roles in manyfundamental cellular events including dorsal/ventral patterning andleft/right axis determination to bone formation and tissue repair. Morerecently, several TGF-β ligands have been shown to be involved in themaintenance or direct the differentiation of stem cells. Due to theirpervasiveness, regulation of TGF-β ligand signaling holds promise forthe treatment of a wide range of different diseases from skeletal andmuscle abnormalities to numerous neoplastic events. Exemplary sequencesare provided herein for various members of this family or proteins,however, one of skill in the art can easily identify homologs andvariants using publicly available databases by word search or sequenceBLAST searches.

There are generally recognized several subfamilies within thesuperfamily of TGF-beta (TGF-β1-(5) as well as the differentiationfactors (e.g., Vg-1), the hormones activin and inhibin, theMullerianinhibiting substance (MIS), osteogenic and morphogenic proteins(e.g., OP-1, OP-2, OP-3, other BMPs), the developmentally regulatedprotein Vgr-1, the growth/differentiation factors (e.g., GDF-1, GDF-3,GDF-9 and dorsalin-1), etc. See, e.g., Spom and Roberts (1990) inPeptide Growth Factors and Their Receptors, Sporn and Roberts, eds.,Springer-Verlag: Berlin pp. 419-472; Weeks and Melton (1987) Cell 51:861-867; Padgett et al. (1987) Nature 325: 81-84; Mason et al. (1985)Nature 318: 659-663; Mason et al. (1987) Growth Factors 1: 77-88; Cateet al. (1986) Cell 45: 685-698; PCT/US90/05903; PCT/US91/07654;PCT/WO94/10202; U.S. Pat. Nos. 4,877,864; 5,141,905; 5,013,649;5,116,738; 5,108,922; 5,106,748; and 5,155,058; Lyons et al. (1989)Proc. Natl. Acad. Sci. USA 86: 4554-58; McPherron et al. (1993) J. Biol.Chem. 268: 3444-3449; Basler et al. (1993) Cell 73: 687-702.

Morphogenic proteins of the TGF-beta superfamily include the mammalianosteogenic protein-1 (OP-1, also known as BMP-7), osteogenic protein-2(OP-2, also known as BMP-8), osteogenic protein-3 (OP3), BMP-2 (alsoknown as BMP-2A or CBMP-2A, and the Drosophila homolog DPP), BMP-3,BMP-4 (also known as BMP-2B or CBMP-2B), BMP-5, BMP-6 and its murinehomolog Vgr-1, BMP-9, BMP-10, BMP-11, BMP-12, GDF3 (also known as Vgr2),GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, BMP-13, BMP-14, BMP-15, GDF-5(also known as CDMP-1 or MP52), GDF-6 (also known as CDMP-2 or BMP13),GDF-7 (also known as CDMP-3 or BMP-12), the Xenopus homolog Vg1 andNODAL, UNIVIN, SCREW, ADMP, NEURAL, etc.

One major roadblock for research involving many of the TGF-β superfamilyligands has been the inability to generate significant quantities of theproteins. While, BMP-2 is known to refold efficiently in vitro, otherTGF-β ligands (activin, Nodal, and BMP-7 for instance) have not shownthe same refolding properties. Other expression systems are available toobtain functional TGF-β ligands. Activin, for example, is expressedusing stably transfected cell lines, such as CHO or transientlytransfected cell lines, such as HEK293 cells.

The largest sub-family of the TGF-β superfamily is the BMP/GDF family,which comprises nearly half of all known ligands. Many of the ligands inBMP/GDF family share both BMP and GDF designations, such as GDF-7 whichalso referred to as BMP-12. In conjunction with being largest family,the BMP/GDF family is also the most extensively studied family. Forexample, x-ray crystal structures have been solved for BMP-2 alone,bound to its type I receptor, or as a ternary complex bound to both itsreceptor types. Additionally, BMP-2, along with BMP-7, has been utilizedas an effective treatment for certain bone injuries. One of the reasonsfor the large amount of structural and therapeutic work involving theBMP/GDF family has been the ability to chemically refold these ligands.Indeed, BMP-2 is one of the most successful TGF-β ligands at refolding,with optimized conditions reported to yield up to 63% active dimer fromstarting material. However, if specific amino acids of BMP-2 could beincorporated into other TGF-β ligands, it may allow for these otherwisenon-refoldable ligands to be refolded opening up the remainder of theTGF-β superfamily to be better studied.

TGF-β ligands are synthesized as inactive precursor molecules composedof an N-terminal pro-domain and a C-terminal mature domain linked by aprotease cleavage site. To be become active, the mature domain must becleaved from the pro-domain, commonly by a convertase, such as furin.Members of the TGF-β superfamily are classified together due to theconserved structural architecture found in their mature domains. Ingeneral, each mature ligand monomer contains 7 cysteines, 6 of whichform three intra-disulfide bonds arranged in a ‘cystine knot’ motif.Stretching outward from the ‘cystine knot’ are 4 beta strands, creating2 curved fingers. The last remaining cysteine forms an inter-disulfidebond with a second ligand monomer, generating a covalently linked dimer.The dimer has the overall appearance of a butterfly with the ‘cystineknot’ as the body and the fingers spreading out like wings. Thefunctional subunit for the TGF-β superfamily is the dimer and they beenshown to exist both as homo- and heterodimers in vivo. Some familymembers, such as GDF-9 and BMP-15, lack the cysteine required to formthe inter-disulfide bond yet they are still able to form stable dimers.

To initiate the signaling process, TGF-β dimers must recruit two sets ofreceptors, termed type I and type II. These receptors areserine/threonine kinases possessing an extracellular domain (ECD)ordered into a three-finger toxin fold, a single transmembrane domain,and a large intracellular kinase domain. TGF-β ligands have been shownto display preferences in their affinity for the different receptortypes. Activin and Nodal exhibit high affinity for type II receptors,while BMP-2 and GDF-5 possess higher affinity for type I receptors.Following the binding of two high affinity receptors to a TGF-β ligand,two lower affinity receptors are then able to bind and join the complex.Upon binding of all four receptors to the TGF-β ligand, forming a6-member ternary complex, the downstream signaling cascade is initiated.The constitutively active type II receptors phosphorylate the type Ireceptors which, in turn, bind and phosphorylate intracellular signalingmolecules called Smads. The Smad molecules then are able to translocateto the nucleus and interact directly with transcriptional regulators.Multiple mechanisms are employed to closely regulate TGF-β signaling atdifferent stages of the cascade: Extracellular antagonists, includingNoggin, follistatin, and Inhibin; pseudo-receptors lacking theintracellular kinase domain, similar to BAMBI; or through intracellularmolecules, such as inhibitory Smads.

TGF-β superfamily shows a high degree of promiscuity by receptors forthe ligands. While there are over 40 TGF-β ligands, there are only 12receptors (7 type 1 and 5 type II). Therefore, receptors must be able tointeract with a multitude of different ligands. For instance, ActRII isknown to bind activin and BMP-7 with high affinity, but binds BMP-2 withmuch lower affinity. In GDF-5, a single amino acid has been found whichdetermines its type I receptor preference, while in BMP-3 a single pointmutation was discovered which alters type II receptor affinity as wellas imparting function to the ligand. The disclosure provides methods tocreate modified TGF-β ligands with novel receptor binding propertiesthereby diversifying TGF-β ligand function as well as compositionshaving such activity.

The disclosure demonstrates a TGF-beta signaling complex by utilizingnovel ligand constructs. Using synthesized chimeric homo- orheterodimeric ligands the ligands the disclosure provides compositionsfor use in dissecting the signaling of TGF-beta family proteins.Furthermore, utilizing such ligands allows a method for distinguishingcontributions of two type I receptor interfaces from each other, and twotype II receptor interfaced each other. The methods and compositions ofthe disclosure demonstrate a correlation between ligand-receptoraffinity, signaling activity, and biological activity. The methods andcompositions of the disclosure shed light on the mechanism andrequirements of the TGF-beta superfamily signaling complex assembly. Inaddition the chimeric ligands provide novel polypeptide for use intreating diseases and disorders associated with TGF-β family ofproteins.

The disclosure provides methods of making and novel chimeric TGF-βligands which possess the ability to be expressed and refolded using,for example, an E. coli or mammalian expression system. These chimeraseither mimic a specific TGF-β ligand's signaling characteristics ordisplay unique signaling properties not seen in nature. In oneembodiment, the disclosure uses activin-βA and BMP-2 as a template togenerate an activin/BMP-2 chimera with the refolding efficiency of BMP-2and the signaling properties of activin-βA; however it should berecognized that any number of TGF-beta protein family members can beused. The chimera design scheme of the disclosure yielded additionalTGF-beta member chimera (e.g., activin/BMP-2) ligands with unnaturalsignaling characteristics and biological activity. Such chimericTGF-beta family polypeptides expand the library of TGF-β ligandsavailable for structural studies as well as facilitate the developmentof novel TGF-β ligands as therapeutic agents.

In one embodiment the disclosure provides a series of activin/BMP-2chimeric ligands which possess unique signaling properties. For example,an activin/BMP-2 ligand of the disclosure exhibited the refoldingcharacteristics of wild type BMP-2 while retaining activin-likesignaling attributes in both in vitro and in vivo studies. Further,‘super’ ligands were generated which are more potent than wild typeBMP-2 and were not inhibited by the BMP antagonist Noggin. Thedisclosure also provides chimeric TGF-beta polypeptides comprising anN-terminus of wild type BMP-2mq operably linked to a different TGF-βligand polypeptide segment. The disclosure demonstrates that theN-terminal portion of wild type BMP-2mq is enough to switch a previouslynon-refolding ligand into a refoldable ligand. These findings highlighta method for obtaining activin and the other TGF-β ligand mimics andindicate how this strategy can be utilized to expand the library ofTGF-β ligands by diversifying their functionality and promote thedevelopment of unnatural ligands for therapeutic purposes.

The nucleic acid sequences and polypeptide sequences of BMP-2 andnaturally occurring variants are known. A wild-type BMP-2 nucleic acidsequence (SEQ ID NO:1) and polypeptide (SEQ ID NO:2) from Rattus sp. areprovided. Met at the position N-terminal to the residue 1 (Q) resultsfrom translation of the bacterial initiation codon (ATG). Furthermoreactivins are also known in the art (see, e.g., SEQ ID NO:5). Thedisclosure provides a number of chimeric TGF-beta family polypeptideshaving at least one N-terminal domain from a BMP-2 and at lease onesecond domain form another TGF-beta family members wherein the chimericpolypeptide display activities different than wild-type parentalproteins.

In one embodiment, two factors were considered when looking to designthe segments of the chimeras. First was a structural consideration. Theoverall TGF-β monomer fold is divided into 6 sections naturally: Betastrand 1 and 2, the pre-helix loop, alpha helix 1, and beta strand 3 and4. The identification and characterization of these subdomains arefurther described in Example 4. The disclosure utilized a chimericstructures to mimic these natural regions in the design. Thus, eachsegment can be indicated by 1, 2, 3, 4, 5, and 6. The secondconsideration was to minimize alterations to the aligned native TGF-betamember sequence during chimera engineering. Therefore, those regionswith sequence identity between the 2 protein sequences were identifiedas putative cross-over points. These regions are suitable for theoverlaps in DNA sequence for PCR strategy and will minimize any changesto the natural sequences. FIG. 7 illustrates the sequence and structureof these considerations. The regions are boxed and numbered according totheir section and are mapped onto the ligand monomer. The areas whichcan be used for the cross-over points as segmental boundaries are shadedas a sequence range in orange. Residue numbering in one embodiment isbased on BMP-2 (SEQ ID NO:2). Thus, cross-over points in generating achimeric polypeptide of the disclosure can be identified by identifyingsimilar structural motifs in combination with at least 60%, 70%, 80%,90%, 95%, 98%, 99% or 100% identity in a segment of the sequence betweenchanges in the structural motif. Cross-overs at these regions (which maybe between 3 to 20 amino acids) minimize disruption of the resultingchimeric polypeptide providing a stabilized chimera.

For example, a chimeric polypeptide comprising the algorithm1b2b3b4b5b6a indicates 6 segments, the letter indicating the parentalstrand of each segment. Thus, in the example “1b2b3b4b5b6a”, segment 1is from parental strand “b” for BMP-2mq, segment 2 is from parentalstrand “b” for BMP-2mq, segment 3 is from parental strand “b” forBMP-2mq, segment 4 is from parental strand “b” for BMP-2mq, segment 5 isfrom parental strand “b” for BMP-2mq, and segment 6 is from parentalstrand “a” for Activin.

In one embodiment, crossover between segments of BMP-2mq and a secondTGF-beta family protein can occur where structural similarity andsequence similary overlap. FIG. 7 depicts such an overlap betweenBMP-2mq and activin, wherein crossovers can be generated between aboutresidue D25-P35, G45-P48; T65-N68; K76-T82; and S88-E94 (residuenumbering is based on BMP-2 (SEQ ID NO:2)). Sequence alignment of BMP-2and activin-βA highlight the boundaries of segments 1 through 6. Activinhas the extra disulfide bond formed between two Cys. Red (or firstshaded boxes on lower sequences) box notes two amino acids of AB2-009swapped into AB2-008. Blue (second shaded box L/Y on lower lines) boxnotes one amino acid changed in Segment 5 of BMP2 for all chimera. Forclarity, BMP-2's Segment 5 contains YYD instead of YLD. Green(KKQ-FFVSFKDI) box denotes a segment introduced into AB2-008 to makeAB2-010, marked as (1a_II) of AB2-010.

FIG. 18 further depicts such crossover regions with reference toadditional members of the TGF-β family or proteins. For example, withreference to FIG. 18, one of skill in the art can see that BMP-3 (SEQ IDNO:43) comprises 110 amino acids. The first vertical line demonstrates ageneral region of cross over and can comprise from 1-5 amino acids oneither side of the vertical line. Accordingly, a first domain from BMP-3can comprise amino acid 1 to about x₁, wherein x₁ is an amino acidcorresponding to residue 20-29 (e.g., x1 is 20, 21, 22, 23, 24, 25, 26,27, 28, or 29). As further shown in FIG. 18, “J1” corresponds toresidues 20-23 of SEQ ID NO:43). J1 refers to a junction region havingconservation across the various species in the TGF-β family of proteins.Accordingly, a first domain of BMP-3 comprises amino acids 1 to aboutx₁, wherein x₁ will be either V or G and the following chimeric domainfrom a second TGF-β family member will begin with either G or W. UsingFIG. 18 as a template one of skill in the art can readily identify thecross-over regions (or junctions points) for the various members of theTGF-β family. It is important to note that not every chimera is requiredto have 6 distinct domains. For example, a cross over at junction 3 (J3)may not be necessary such that only 5 or fewer domains from distinctfamily member are present in the final chimera.

Other methods for identifying crossover locations may be employed in thegeneration of chimeric TGF-beta family polypeptides. For example, SCHEMAis a computational based method for predicting which fragments ofhomologous proteins can be recombined without affecting the structuralintegrity of the protein (see, e.g., Meyer et al., (2003) Protein Sci.,12:1686-1693). Chimeras with higher stability are identifiable bydetermining the additive contribution of each segment to the overallstability, either by use of linear regression of sequence-stabilitydata, or by reliance on consensus analysis of the MSAs of folded versusunfolded proteins. SCHEMA recombination ensures that the chimeras retainbiological function and exhibit high sequence diversity by conservingimportant functional residues while exchanging tolerant ones.

As presented in this disclosure, it has been found that when theserecombined, functional chimeric TGF-beta family polypeptides aregenerated their ligand specificity can be improved or biologicalactivity can be altered or improved compared to a unrecombined parentalpolypeptide. Because of differences in activity/ligand profiles, theseengineered chimeric TGF-beta family polypeptides provide a unique basisto screen for activities for ligand specific activation and inhibition,provide novel therapeutic polypeptides and research reagents.

For example, in the chimeras of the disclosure, domain 1, 2, 3, 4, 5,and 6 can be selected from the following sequences (Table A) wherein thepolypeptide comprises a structure (domain 1-domain 2-domain 3-domain4-domain 5-domain 6):

TABLE A Amino acids (domain #) SEQ ID NO: Variable definition 1-x₁ 2 x₁is 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 (1) 1-x₁ 5 x₁ is 22,23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 (1) 1-x₁ 43 x₁ is 20, 21, 22,23, 24, 25, 26, 27, 28, or 29 (1) 1-x₁ 45 x₁ is 27, 28, 29, 30, 31, 32,33, 34, 35, or 36 (1) 1-x₁ 47 x₁ is 43, 44, 44, 46 47, 48, 49, 50, 51,or 52 (1) 1-x₁ 49 x₁ is 43, 44, 44, 46 47, 48, 49, 50, 51, or 52 (1)1-x₁ 51 x₁ is 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 (1) 1-x₁ 53x₁ is 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 (1) 1-x₁ 55 x₁ is19, 20, 21, 22, 23, 24, 25, 26 27, or 28 (1) 1-x₁ 57 x₁ is 18, 19, 20,21, 22, 23, 24, 25, 26 or 27 (1) 1-x₁ 59 x₁ is 36, 37, 38, 29, 40, 41,42, 43, 44, or 45 (1) 1-x₁ 61 x₁ is 25, 26, 27, 28, 29, 30, 31, 32, 33or 34 (1) 1-x₁ 63 x₁ is 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 (1)1-x₁ 65 x₁ is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 (1) 1-x₁ 67x₁ is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 (1) 1-x₁ 69 x₁ is 40,41, 42, 43, 44, 45, 46, 47, 48 or 49 (1) 1-x₁ 71 x₁ is 25, 26, 27, 28,29, 30, 31, 32, 33 or 34 (1) 1-x₁ 73 x₁ is 45, 46, 47, 48, 49, 50, 51,52, 53, 54, or 55 (1) 1-x₁ 75 x₁ is 28, 29, 30, 31, 32, 33, 34, 35, 36,or 37 (1) 1-x₁ 77 x₁ is 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 (1)1-x₁ 79 x₁ is 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 (1) 1-x₁ 81 x₁is 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 (1) 1-x₁ 83 x₁ is 22, 23,24, 25, 26, 27, 28, 29, 30 or 31 (1) 1-x₁ 85 x₁ is 22, 23, 24, 25, 26,27, 28, 29, 30 or 31 (1) 1-x₁ 87 x₁ is 22, 23, 24, 25, 26, 27, 28, 29,30 or 31 (1) 1-x₁ 89 x₁ is 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 (1)1-x₁ 91 x₁ is 42, 43, 44, 45, 46, 47, 48, 49, 50 or 51 (1) 1-x₁ 93 x₁ is28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 (1) 1-x₁ 95 x₁ is 24, 25, 26,27, 28, 29, 30, 31, 32 or 33 (1) 1-x₁ 97 x₁ is 24, 25, 26, 27, 28, 29,30, 31, 32 or 33 (1) x₁ - x₂ 2 x₁ is 25, 26, 27, 28, 29, 30, 31, 32, 33,34 or 35 (2) x₂ is 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 x₁ - x₂ 5x₁ is 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 (2) x₂ is 41, 42, 43,44, 45, 46, 47, 48, 49, 50 or 51 x₁ - x₂ 43 x₁ is 20, 21, 22, 23, 24,25, 26, 27, 28 or 29 (2) x₂ is 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47x₁ - x₂ 45 x₁ is 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 (2) x₂ is 46,47, 48, 49, 50, 51, 52, 53, 54 or 55 x₁ - x₂ 47 x₁ is 43, 44, 44, 46 47,48, 49, 50, 51 or 52 (2) x₂ is 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70x₁ - x₂ 49 x₁ is 43, 44, 44, 46 47, 48, 49, 50, 51 or 52 (2) x₂ is 61,62, 63, 64, 65, 66, 67, 68, 69 or 70 x₁ - x₂ 51 x₁ is 50, 51, 52, 53,54, 55, 56, 57, 58, 59 or 60 (2) x₂ is 68, 69, 70, 71, 72, 73, 74, 75,76, 77 or 78 x₁ - x₂ 53 x₁ is 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or60 (2) x₂ is 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 or 78 x₁ - x₂ 55 x₁is 19, 20, 21, 22, 23, 24, 25, 26 27, or 28 (2) x₂ is 38, 39, 40, 41,42, 43, 44, 45, 46 or 47 x₁ - x₂ 57 x₁ is 18, 19, 20, 21, 22, 23, 24,25, 26 or 27 (2) x₂ is 37, 38, 39, 40, 41, 42, 43, 44, 45 or 49 x₁ - x₂59 x₁ is 36, 37, 38, 29, 40, 41, 42, 43, 44 or 45 (2) x₂ is 53, 54, 55,56, 57, 58, 59, 60, 61 or 62 x₁ - x₂ 61 x₁ is 25, 26, 27, 28, 29, 30,31, 32, 33 or 34 (2) x₂ is 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53x₁ - x₂ 63 x₁ is 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 (2) x₂ is 43,44, 45, 46, 47, 48, 49, 50, 51, 52 or 53 x₁ - x₂ 65 x₁ is 30, 31, 32,33, 34, 35, 36, 37, 38, 39 or 40 (2) x₂ is 48, 49, 50, 51, 52, 53, 54,55, 56, 57 or 58 x₁ - x₂ 67 x₁ is 30, 31, 32, 33, 34, 35, 36, 37, 38, 39or 40 (2) x₂ is 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58 x₁ - x₂ 69x₁ is 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 (2) x₂ is 57, 58, 59, 60,61, 62, 63, 64, 65 ot 66 x₁ - x₂ 71 x₁ is 25, 26, 27, 28, 29, 30, 31,32, 33 or 34 (2) x₂ is 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 x₁ - x₂73 x₁ is 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 (2) x₂ is 63, 64,65, 66, 67, 68, 69, 70, 71, 72 or 73 x₁ - x₂ 75 x₁ is 28, 29, 30, 31,32, 33, 34, 35, 36, or 37 (2) x₂ is 46, 47, 48, 49, 50, 51, 52, 53, 54or 55 x₁ - x₂ 77 x₁ is 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 (2) x₂is 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53 x₁ - x₂ 79 x₁ is 24, 25,26, 27, 28, 29, 30, 31, 32 or 33 (2) x₂ is 44, 45, 46, 47, 48, 49, 50,51, 52 or 53 x₁ - x₂ 81 x₁ is 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30(2) x₂ is 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 x₁ - x₂ 83 x₁ is 22,23, 24, 25, 26, 27, 28, 29, 30 or 31 (2) x₂ is 41, 42, 43, 44, 45, 46,47, 48, 49, 50 or 51 x₁ - x₂ 85 x₁ is 22, 23, 24, 25, 26, 27, 28, 29, 30or 31 (2) x₂ is 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 51 x₁ - x₂ 87x₁ is 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 (2) x₂ is 41, 42, 43, 44,45, 46, 47, 48, 49, 50 or 51 x₁ - x₂ 89 x₁ is 22, 23, 24, 25, 26, 27,28, 29, 30 or 31 (2) x₂ is 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 51x₁ - x₂ 91 x₁ is 42, 43, 44, 45, 46, 47, 48, 49, 50 or 51 (2) x₂ is 59,60, 61, 62, 63, 65, 65, 66, 67 or 68 x₁ - x₂ 93 x₁ is 28, 29, 30, 31,32, 33, 34, 35, 36 or 37 (2) x₂ is 46, 47, 48, 49, 50, 51, 52, 53, 54 or55 x₁ - x₂ 95 x₁ is 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 (2) x₂ is44, 45, 46, 47, 48, 49, 50, 51, 52 or 53 x₁ - x₂ 97 x₁ is 24, 25, 26,27, 28, 29, 30, 31, 32 or 33 (2) x₂ is 44, 45, 46, 47, 48, 49, 50, 51,52 or 53 x₂ - x₃ 2 x₂ is 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 (3)x₃ is 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 x₂ - x₃ 5 x₂is 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 51 (3) x₃ is 55, 56, 57,58, 59, 60, 61, 62, 63, 6, 4 65, 66, 67, 68, 69, 70, 71, 72, 73, or 74x₂ - x₃ 43 x₂ is 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47 (3) x₃ is 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 x₂ - x₃ 45 x₂ is46, 47, 48, 49, 50, 51, 52, 53, 54 or 55 (3) x₃ is 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73 or 74 x₂ - x₃ 47 x₂ is 61, 62, 63, 64,65, 66, 67, 68, 69 or 70 (3) x₃ is 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87 or 88 x₂ - x₃ 49 x₂ is 61, 62, 63, 64, 65, 66, 67, 68, 69or 70 (3) x₃ is 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87 or 88x₂ - x₃ 51 x₂ is 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 or 78 (3) x₃ is82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95 x₂ - x₃ 53 x₂is 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 or 78 (3) x₃ is 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95 x₂ - x₃ 55 x₂ is 38, 39,40, 41, 42, 43, 44, 45, 46 or 47 (3) x₃ is 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64 or 65 x₂ - x₃ 57 x₂ is 37, 38, 39, 40, 41, 42,43, 44, 45 or 49 (3) x₃ is 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, or 64 x₂ - x₃ 59 x₂ is 53, 54, 55, 56, 57, 58, 59, 60, 61 or 62(3) x₃ is 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 or 81 x₂ -x₃ 61 x₂ is 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53 (3) x₃ is 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75or 76 x₂ - x₃ 63 x₂ is 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53 (3)x₃ is 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 x₂ - x₃ 65 x₂is 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58 (3) x₃ is 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74 or 75 x₂ - x₃ 67 x₂ is 48, 49, 50,51, 52, 53, 54, 55, 56, 57 or 58 (3) x₃ is 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74 or 75 x₂ - x₃ 69 x₂ is 57, 58, 59, 60, 61, 62, 63,64, 65 ot 66 (3) x₃ is 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 or84 x₂ - x₃ 71 x₂ is 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 (3) x₃ is53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 x₂ - x₃ 73 x₂ is63, 64, 65, 66, 67, 68, 69, 70, 71, 72 or 73 (3) x₃ is 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89 or 90 x₂ - x₃ 75 x₂ is 46, 47, 48, 49,50, 51, 52, 53, 54 or 55 (3) x₃ is 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72 or 73 x₂ - x₃ 77 x₂ is 43, 44, 45, 46, 47, 48, 49, 50, 51, 52or 53 (3) x₃ is 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 x₂ - x₃79 x₂ is 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53 (3) x₃ is 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67 or 68 x₂ - x₃ 81 x₂ is 39, 40, 41,42, 43, 44, 45, 46, 47 or 48 (3) x₃ is 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65 or 66 x₂ - x₃ 83 x₂ is 41, 42, 43, 44, 45, 46, 47, 48,49, 50 or 51 (3) x₃ is 55, 56, 5, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, or 74 x₂ - x₃ 85 x₂ is 41, 42, 43, 44, 45,46, 47, 48, 49, 50 or 51 (3) x₃ is 55, 56, 5, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, or 74 x₂ - x₃ 87 x₂ is 41, 42,43, 44, 45, 46, 47, 48, 49, 50 or 51 (3) x₃ is 55, 56, 5, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, or 74 x₂ - x₃ 89 x₂is 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 51 (3) x₃ is 55, 56, 5, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, or 74 x₂ -x₃ 91 x₂ is 59, 60, 61, 62, 63, 65, 65, 66, 67 or 68 (3) x₃ is 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 or 87 x₂ - x₃ 93 x₂ is 46,47, 48, 49, 50, 51, 52, 53, 54 or 55 (3) x₃ is 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69 or 70 x₂ - x₃ 95 x₂ is 44, 45, 46, 47, 48, 49,50, 51, 52 or 53 (3) x₃ is 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or65 x₂ - x₃ 97 x₂ is 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53 (3) x₃ is54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 x₃ - x₄ 2 x₃ is 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 (4) x₄ is 78, 79, 80, 81,82, 83, 84 or 85 x₃ - x₄ 5 x₃ is 55, 56, 57, 58, 59, 60, 61, 62, 63, 6,4 65, 66, 67, 68, (4) 69, 70, 71, 72, 73, or 74 x₄ is 79, 80, 81, 82,83, 84, 85 or 86 x₃ - x₄ 43 x₃ is 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64 or 65 (4) x₄ is 73, 74, 75, 76, 77, 78, 79 or 80 x₃ - x₄45 x₃ is 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 or 74 (4) x₄is 79, 80, 81, 82, 83, 84, 85 or 86 x₃ - x₄ 47 x₃ is 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87 or 88 (4) x₄ is 95, 96, 97, 98, 99, 100,101, 102 or 103 x₃ - x₄ 49 x₃ is 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87 or 88 (4) x₄ is 95, 96, 97, 98, 99, 100, 101, 102 or 103 x₃ -x₄ 51 x₃ is 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95 (4)x₄ is 102, 103, 104, 105, 106, 107, 108, 109 or 110 x₃ - x₄ 53 x₃ is 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95 (4) x₄ is 102, 103,104, 105, 106, 107, 108, 109 or 110 x₃ - x₄ 55 x₃ is 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64 or 65 (4) x₄ is 72, 73, 74, 75, 76, 77,78, 79 or 80 x₃ - x₄ 57 x₃ is 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, or 64 (4) x₄ is 71, 72, 73, 74, 75, 76, 77, 78 or 79 x₃ - x₄59 x₃ is 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 or 81 (4) x₄is 78, 79, 80, 81, 82, 83, 84, 85 or 86 x₃ - x₄ 61 x₃ is 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, (4) 71, 72, 73, 74, 75 or 76 x₄is 82, 83, 84, 85, 86, 87, 88, 89 or 90 x₃ - x₄ 63 x₃ is 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69 or 70 (4) x₄ is 77, 78, 79, 80, 81, 82,83, 84 or 85 x₃ - x₄ 65 x₃ is 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74 or 75 (4) x₄ is 83, 84, 85, 86, 87, 88, 89, 90 or 91 x₃ - x₄ 67x₃ is 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75 (4) x₄ is 83,84, 85, 86, 87, 88, 89, 90 or 91 x₃ - x₄ 69 x₃ is 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83 or 84 (4) x₄ is 92, 93, 94, 95, 96, 97, 98,99 or 100 x₃ - x₄ 71 x₃ is 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64 or 65 (4) x₄ is 72, 73, 74, 75, 76, 77, 78, 79 or 80 x₃ - x₄ 73 x₃ is78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90 (4) x₄ is 98, 99,100, 101, 102, 103, 104, 105 or 106 x₃ - x₄ 75 x₃ is 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72 or 73 (4) x₄ is 82, 83, 84, 85, 86, 87, 88,89, or 90 x₃ - x₄ 77 x₃ is 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or65 (4) x₄ is 72, 73, 74, 75, 76, 77, 78, 79 or 80 x₃ - x₄ 79 x₃ is 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 or 68 (4) x₄ is 76, 77, 78,79, 80, 81, 82, 83 or 84 x₃ - x₄ 81 x₃ is 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65 or 66 (4) x₄ is 74, 75, 76, 77, 78, 79, 80, 81 or 82x₃ - x₄ 83 x₃ is 55, 56, 5, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, (4) 70, 71, 72, 73, or 74 x₄ is 79, 80, 81, 82, 83, 84, 85, 86 or 87x₃ - x₄ 85 x₃ is 55, 56, 5, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, (4) 70, 71, 72, 73, or 74 x₄ is 78, 79, 80, 81, 82, 83, 84, 85 or 86x₃ - x₄ 87 x₃ is 55, 56, 5, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, (4) 70, 71, 72, 73, or 74 x₄ is 79, 80, 81, 82, 83, 84, 85, 86 or 87x₃ - x₄ 89 x₃ is 55, 56, 5, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, (4) 70, 71, 72, 73, or 74 x₄ is 77, 78, 79, 80, 81, 82, 83, 84 or 85x₃ - x₄ 91 x₃ is 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 or87 (4) x₄ is 96, 97, 98, 99, 100, 101, 102, 103 or 104 x₃ - x₄ 93 x₃ is58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 (4) x₄ is 77, 78,79, 80, 81, 82, 83, 84, or 85 x₃ - x₄ 95 x₃ is 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64 or 65 (4) x₄ is 76, 77, 78, 79, 80, 81, 82, 83 or 84x₃ - x₄ 97 x₃ is 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 (4) x₄is 76, 77, 78, 79, 80, 81, 82, 83 or 84 x₄ - x₅ 2 x₄ is 78, 79, 80, 81,82, 83, 84 or 85 (5) x₅ is 89, 90, 91, 92, 93, 94, 95, 96, 97 o r 98x₄ - x₅ 5 x₄ is 79, 80, 81, 82, 83, 84, 85 or 86 (5) x₅ is 91, 92, 93,94, 95, 96, 97, 98, 99 or 100 x₄ - x₅ 43 x₄ is 73, 74, 75, 76, 77, 78,79 or 80 (5) x₅ is 85, 86, 87, 88, 89, 90 91, 92 or 93 x₄ - x₅ 45 x₄ is79, 80, 81, 82, 83, 84, 85 or 86 (5) x₅ is 91, 92, 93, 94, 95, 96, 97,98, 99 or 100 x₄ - x₅ 47 x₄ is 95, 96, 97, 98, 99, 100, 101, 102 or 103(5) x₅ is 107, 108, 109, 110, 111, 112, 113, 114 or 115 x₄ - x₅ 49 x₄ is95, 96, 97, 98, 99, 100, 101, 102 or 103 (5) x₅ is 107, 108, 109, 110,111, 112, 113, 114 or 115 x₄ - x₅ 51 x₄ is 102, 103, 104, 105, 106, 107,108, 109 or 110 (5) x₅ is 114, 115, 116, 117, 118, 119, 120, 121, or 122x₄ - x₅ 53 x₄ is 102, 103, 104, 105, 106, 107, 108, 109 or 110 (5) x₅ is114, 115, 116, 117, 118, 119, 120, 121, or 122 x₄ - x₅ 55 x₄ is 72, 73,74, 75, 76, 77, 78, 79 or 80 (5) x₅ is 84, 85, 86, 87, 88, 89, 90, 91,92 or 93 x₄ - x₅ 57 x₄ is 71, 72, 73, 74, 75, 76, 77, 78 or 79 (5) x₅ is83, 84, 85, 86, 87, 88, 89, 90, 91, or 92 x₄ - x₅ 59 x₄ is 78, 79, 80,81, 82, 83, 84, 85 or 86 (5) x₅ is 100, 101, 102, 103, 104, 106, 106,107 or 108 x₄ - x₅ 61 x₄ is 82, 83, 84, 85, 86, 87, 88, 89 or 90 (5) x₅is 94, 95, 96, 97, 98, 99, 100, 101 or 102 x₄ - x₅ 63 x₄ is 77, 78, 79,80, 81, 82, 83, 84 or 85 (5) x₅ is 89, 90, 91, 92, 93, 94, 95, 96, 97 or 98 x₄ - x₅ 65 x₄ is 83, 84, 85, 86, 87, 88, 89, 90 or 91 (5) x₅ is 95,96, 97, 98, 99, 100, 101, 102 or 103 x₄ - x₅ 67 x₄ is 83, 84, 85, 86,87, 88, 89, 90 or 91 (5) x₅ is 95, 96, 97, 98, 99, 100, 101, 102 or 103x₄ - x₅ 69 x₄ is 92, 93, 94, 95, 96, 97, 98, 99 or 100 (5) x₅ is 104,105, 106, 107, 108, 109, 110, 111 or 112 x₄ - x₅ 71 x₄ is 72, 73, 74,75, 76, 77, 78, 79 or 80 (5) x₅ is 84, 85, 86, 87, 88, 89, 90, 91, 92 or93 x₄ - x₅ 73 x₄ is 98, 99, 100, 101, 102, 103, 104, 105 or 106 (5) x₅is 110, 111, 112, 113, 114, 115, 116, 117 or 118 x₄ - x₅ 75 x₄ is 82,83, 84, 85, 86, 87, 88, 89, or 90 (5) x₅ is 94, 95, 96, 97, 98, 99, 100,101 or 102 x₄ - x₅ 77 x₄ is 72, 73, 74, 75, 76, 77, 78, 79 or 80 (5) x₅is 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 x₄ - x₅ 79 x₄ is 76, 77, 78,79, 80, 81, 82, 83 or 84 (5) x₅ is 88, 89, 90, 91, 92, 93, 94, 95 or 96x₄ - x₅ 81 x₄ is 74, 75, 76, 77, 78, 79, 80, 81 or 82 (5) x₅ is 86, 87,88, 89, 90, 91, 92, 93 or 94 x₄ - x₅ 83 x₄ is 79, 80, 81, 82, 83, 84,85, 86 or 87 (5) x₅ is 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 x₄ - x₅85 x₄ is 78, 79, 80, 81, 82, 83, 84, 85 or 86 (5) x₅ is 90, 91, 92, 93,94, 95, 96, 97, 98 or 99 x₄ - x₅ 87 x₄ is 79, 80, 81, 82, 83, 84, 85, 86or 87 (5) x₅ is 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 x₄ - x₅ 89 x₄is 77, 78, 79, 80, 81, 82, 83, 84 or 85 (5) x₅ is 89, 90, 91, 92, 93,94, 95, 96, 97 o r 98 x₄ - x₅ 91 x₄ is 96, 97, 98, 99, 100, 101, 102,103 or 104 (5) x₅ is 108, 109, 110, 111, 112, 113, 114, 115 or 116 x₄ -x₅ 93 x₄ is 77, 78, 79, 80, 81, 82, 83, 84, or 85 (5) x₅ is 89, 90, 91,92, 93, 94, 95, 96, 97 o r 98 x₄ - x₅ 95 x₄ is 76, 77, 78, 79, 80, 81,82, 83 or 84 (5) x₅ is 88, 89, 90, 91, 92, 93, 94, 95 or 96 x₄ - x₅ 97x₄ is 76, 77, 78, 79, 80, 81, 82, 83 or 84 (5) x₅ is 88, 89, 90, 91, 92,93, 94, 95 or 96 x₅ - 114 2 x₅ is 89, 90, 91, 92, 93, 94, 95, 96, 97 o r98 (6) x₅ - 116 5 x₅ is 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 (6)x₅ - 110 43 x₅ is 85, 86, 87, 88, 89, 90 91, 92 or 93 (6) x₅ - 116 45 x₅is 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 (6) x₅ - 132 47 x₅ is 107,108, 109, 110, 111, 112, 113, 114 or 115 (6) x₅ - 132 49 x₅ is 107, 108,109, 110, 111, 112, 113, 114 or 115 (6) x₅ - 139 51 x₅ is 114, 115, 116,117, 118, 119, 120, 121, or 122 (6) x₅ - 139 53 x₅ is 114, 115, 116,117, 118, 119, 120, 121, or 122 (6) X₅ - 110 55 x₅ is 84, 85, 86, 87,88, 89, 90, 91, 92 or 93 (6) x₅ - 108 57 x₅ is 83, 84, 85, 86, 87, 88,89, 90, 91, or 92 (6) x₅ - 125 59 x₅ is 100, 101, 102, 103, 104, 106,106, 107 or 108 (6) x₅ - 119 61 x₅ is 94, 95, 96, 97, 98, 99, 100, 101or 102 (6) x₅ - 114 63 x₅ is 89, 90, 91, 92, 93, 94, 95, 96, 97 o r 98(6) x₅ - 120 65 x₅ is 95, 96, 97, 98, 99, 100, 101, 102 or 103 (6) x₅ -120 67 x₅ is 95, 96, 97, 98, 99, 100, 101, 102 or 103 (6) x₅ - 129 69 x₅is 104, 105, 106, 107, 108, 109, 110, 111 or 112 (6) x₅ - 109 71 x₅ is84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 (6) x₅ - 135 73 x₅ is 110, 111,112, 113, 114, 115, 116, 117 or 118 (6) x₅ - 119 75 x₅ is 94, 95, 96,97, 98, 99, 100, 101 or 102 (6) x₅ - 109 77 x₅ is 84, 85, 86, 87, 88,89, 90, 91, 92 or 93 (6) x₅ - 113 79 x₅ is 88, 89, 90, 91, 92, 93, 94,95 or 96 (6) x₅ - 110 81 x₅ is 86, 87, 88, 89, 90, 91, 92, 93 or 94 (6)x₅ - 116 83 x₅ is 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 (6) x₅ - 11585 x₅ is 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 (6) x₅ - 116 87 x₅ is91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 (6) x₅ - 114 89 x₅ is 89, 90,91, 92, 93, 94, 95, 96, 97 o r 98 (6) x₅ - 134 91 x₅ is 108, 109, 110,111, 112, 113, 114, 115 or 116 (6) x₅ - 113 93 x₅ is 89, 90, 91, 92, 93,94, 95, 96, 97 o r 98 (6) x₅ - 112 95 x₅ is 88, 89, 90, 91, 92, 93, 94,95 or 96 (6) x₅ - 112 97 x₅ is 88, 89, 90, 91, 92, 93, 94, 95 or 96 (6)

In some embodiments, domain 3 may be derived from the same parent aseither domain 2, domain 4 or both domain 3 and 4.

In some embodiment, J1 (Junction 1) between domain 1 and domain 2comprises the consensus sequence Z₁Z₂W, wherein Z₁ is selected from thegroup L, V, F, and M, and Z₂ is G or K, wherein 2 of the 3 amino acidsare found at the C-terminus of the first domain or the N-terminus of thesecond domain. In some embodiment, J2 (Junction 2) between domain 2 anddomain 3 comprises the consensus sequence CZ₁G, wherein Z₁ is selectedfrom the group H, S, A, L, I, E, K, Q and D, wherein 2 of the 3 aminoacids are found at the C-terminus of the second domain or the N-terminusof the third domain. In some embodiment, J3 (Junction 3) between domain3 and domain 4 comprises the consensus sequence Z₁Z₂Z₃, wherein Z₁ isselected from the group T, S, P, G and I, Z₂ is selected from the groupconsisting of N, K, V, M, H and Y, and Z₃ is selected from the groupconsisting of H, Y, S, T and P, wherein 2 of the 3 amino acids are foundat the C-terminus of the third domain or the N-terminus of the fourthdomain. In some embodiment, J4 (Junction 4) between domain 4 and domain5 comprises the consensus sequence Z₁CZ₂, wherein Z₁ is selected fromthe group C, S and V, and Z₂ is selected from the group consisting of V,A, I and T, wherein 2 of the 3 amino acids are found at the C-terminusof the fourth domain or the N-terminus of the Fifth domain. In someembodiment, J5 (Junction 1) between the domain 5 and domain 6 comprisesthe consensus sequence Z₁Z₂Z₃, wherein Z₁ is selected from the group L,R and V, Z₂ is selected from the group consisting of T, Q, Y, F and M,and Z₃ is selected from the group consisting of L, F, Y, K, I, Q, V andT, wherein 2 of the 3 amino acids are found at the C-terminus of thefifth domain or the N-terminus of the sixth domain.

In one embodiment, the disclosure provides the following domains (TableB) for reach of the TGF-beta family members that may be recombined toform a chimera of the disclosure having increased or improved biologicalactivity (e.g., resistance to inactivation and the like).

TABLE B Domain Domain Domain Domain Domain Domain 1 2 3 4 5 6 BMP-2 1-3031-48 49-68 69-81 82-93 94-114 BMP-3 1-24 25-42 43-62 63-77 78-89 90-110BMP-4 1-32 33-50 51-70 71-83 84-95 96-116 BMP-5 1-47 48-65 66-85 86-99100-111 112-132  BMP-6 1-47 48-65 66-85 86-99 100-111 112-132  BMP-71-54 55-72 73-92  93-106 107-118 119-139  BMP-8 1-54 55-72 73-92  93-106107-118 119-139  BMP-9 1-24 25-42 43-62 63-76 77-88 89-110 BMP-10 1-2324-41 42-61 62-75 76-87 88-108 BMP-15 1-40 41-58 59-78 79-92  93-104105-125  GDF-1 1-30 31-48 49-72 73-86 87-98 99-119 GDF-3 1-30 31-4849-68 69-81 82-93 94-114 GDF-5 1-35 36-53 54-73 74-87 88-99 100-120 GDF-6 1-35 36-53 54-73 74-87 88-99 100-120  GDF-7 1-44 45-62 63-82 83-9697-108 109-129  GDF-8 1-30 31-48 49-63 64-76 77-88 89-109 GDF-9 1-5051-68 69-88  89-102 103-114 115-135  GDF-10 1-33 34-51 52-71 72-86 87-9899-119 GDF-11 1-30 31-48 49-63 64-76 77-88 89-109 GDF-15 1-31 32-4950-66 67-80 81-92 93-112 NODAL 1-26 27-44 45-64 65-78 79-90 91-110ACTIVIN-A 1-27 28-45 46-68 69-83 84-95 96-116 Activin-B 1-27 28-45 46-6869-82 83-94 95-115 Activin-C 1-27 28-45 46-68 69-83 84-95 96-116Activin-E 1-27 28-45 46-68 69-81 82-93 94-114 INHIBIN-A 1-46 47-64 65-84 85-100 101-112 113-134  TGF-beta1 1-32 33-50 51-68 69-81 82-93 94-113TGF-beta2 1-31 32-49 50-67 68-80 81-92 93-112 TGF-beta3 1-31 32-49 50-6768-80 81-92 93-112

Thus, as illustrated by various embodiments herein, the disclosureprovides chimeric TGF-beta family polypeptides, wherein a first TGF-betafamily protein (i.e., a first parental protein) is recombined with asecond different TGF-beta family protein to provide a chimericpolypeptide. Table 2, below, provides exemplary chimeric polypeptides ofthe disclosure. In some embodiments, the polypeptide comprises one ormore domains of a BMP-2 protein, wherein the segments of the BMP-2protein comprise segment 1: amino acid residue from about 1 to about x₁of SEQ ID NO:2 (“1b”); segment 2 is from about amino acid residue x₁ toabout x₂ of SEQ ID NO:2 (“2b”); segment 3 is from about amino acidresidue x₂ to about x₃ of SEQ ID NO:2 (“3b”); segment 4 is from aboutamino acid residue x₃ to about x₄ of SEQ ID NO:2 (“4b”); segment 5 isfrom about amino acid residue x₄ to about x₅ of SEQ ID NO:2 (“5b”); andsegment 6 is from about amino acid residue x₅ to about x₆ of SEQ ID NO:2(“6b”); and wherein: x₁ is residue 25, 26, 27, 28, 29, 30, 31, 32, 33,34, or 35 of SEQ ID NO:2; x₂ is residue 45, 46, 47, or 48 of SEQ IDNO:2; x₃ is residue 65, 66, 67, or 68 of SEQ ID NO:2; x₄ is residue 76,77, 78, 79, 80, 81 or 82 of SEQ ID NO:2; x₅ is residue 88, 89, 90, 91,92, 93, or 94 of SEQ ID NO:2; and x₆ is residue 112, 113, or 114 or SEQID NO:2, corresponding to the C-terminus of BMP-2, such that acontinguous polypeptide comprising segments 1b2b3b4b5b6b comprises awild-type BMP-2 following the translation initiation codon (ATG).Homologs and proteins having at least about 80%, 90%, 95%, 98%, and 99%identity to the foregoing sequences are also included by the disclosure.

In other embodiments, the polypeptide comprises one or more domains ofan activin protein, wherein the segments of the activin protein comprisesegment 1: amino acid residue from about 1 to about x₁ of SEQ ID NO:5(“1a”); segment 2 is from about amino acid residue x₁ to about x₂ of SEQID NO:5 (“2a”); segment 3 is from about amino acid residue x₂ to aboutx₃ of SEQ ID NO:5 (“3a”); segment 4 is from about amino acid residue x₃to about x₄ of SEQ ID NO:5 (“4a”); segment 5 is from about amino acidresidue x₄ to about x₅ of SEQ ID NO:5 (“5a”); and segment 6 is fromabout amino acid residue x₅ to about x₆ of SEQ ID NO:5 (“6a”); andwherein: x₁ is residue 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 ofSEQ ID NO:5; x₂ is residue 42, 43, 44, or 45 of SEQ ID NO:5; x₃ isresidue 61, 62, 63, or 64 of SEQ ID NO:5; x₄ is residue 78, 79, 80, 81,82, 83 or 84 of SEQ ID NO:5; x₅ is residue 90, 91, 92, 93, 94, 95 or 96of SEQ ID NO:5; and x₆ is residue 114, 115, or 116 or SEQ ID NO:5,corresponding to the C-terminus of activin, such that a continguouspolypeptide comprising segments 1a2a3a4a5a6a comprises a wild-typemature activin protein. Homologs and proteins having at least about 80%,90%, 95%, 98%, and 99% identity to the foregoing sequences are alsoincluded by the disclosure.

In some embodiments, chimeric TGF-beta family polypeptide has a chimericsegmental structure selected from the group consisting of: 1b2b3b4b5b6b;1b2b3b4b5b6a; 1b2b3b4b5a6a; 1b2b3b4b5a6b; 1b2b3a4a5a6a; 1b2b3a4a5b6a;1b2a3a4a5a6a; 1b2a3a4a5a6a L66V/V67I; 1b(1a_II)2a3a4a5a6a; 1b2a3a4a5a6b;1b2a3a4a5b6b; 1b2a3a4a5b6a; 1b2a3b4b5b6a; 1b2a3b4b5a6a; and1b2a3b4b5a6b.

In other embodiment, the chimeric polypeptide may be fused to anadditional heterologous polypeptide to generate a chimeric fusionpolypeptide. The heterologous polypeptide may be, for example, a peptideuseful for purification or that permits oligomerization of multiplechimeric polypeptides of the disclosure. The heterologous may bechemically conjugated to the chimeric polypeptide or may be operablylinked in-frame with a coding sequence for the chimeric polypeptide.

In more particular embodiments, the polypeptide comprises a sequencethat is (a) at least 80%, 90%, 95%, 98%, or 99% identical to sequenceselected from the group consisting of SEQ ID NO: 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, and 33 and has BMP-2 activity; (b) comprisesa sequence as set forth in SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, and 33; (c) is encoded by a polynucleotide comprising asequence selected from the group consisting of SEQ ID NO:6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, and 32; or (d) comprises a sequencedescribed by an algorithm selected from the group consisting of1b2b3b4b5b6b; 1b2b3b4b5b6a; 1b2b3b4b56a; 1b2b3b4b5a6b; 1b2b3b4a5a6a;1b2b3b4a5b6b; 1b2b3b4a5a6b; 1b2b3b4a5b6a; 1b2b3a4a5a6a; 1b2b3a4a5a6b;1b2b3a4a5b6b; 1b2b3a4a5b6a; 1b2b3a4b5b6b; 1b2b3a4b5b6a; 1b2b3a4b5a6a;1b2b3a4b5a6b; 1b2a3a4a5a6a; 1b2a3a4a5a6b; 1b2a3a4a5b6b; 1b2a3a4a5b6a;1b2a3a4b5b6b; 1b2a3a4b5b6a; 1b2a3a4b5a6b; 1b2a3a4b5a6a; 1b2a3b4b5b6b;1b2a3b4b5b6a; 1b2a3b4b5a6a; 1b2a3b4b5a6b; 1b2a3b4a5a6a; 1b2a3b4a5b6a;1b2a3b4a5b6b; 1b2a3b4a5a6b; 1b2a3a4a5a6a L66V/V67I; and1b(1a_II)2a3a4a5a6a. In yet another embodiment, the disclosure providesa chimeric TGF-beta polypeptide comprising a segment from BMP-2 andsegments from BMP-7 (e.g., a 1b-BMP7 polypeptide; see, e.g., SEQ IDNO:35). In yet another embodiment, the disclosure provides a chimericTGF-beta polypeptide comprising a segment from BMP-2 and segments fromBMP-9 (e.g., a 1b-BMP9; see, e.g., SEQ ID NO:37). In yet anotherembodiment, the disclosure provides a chimeric TGF-beta polypeptidecomprising a segment from BMP-2 and segments from GDF-7 (e.g., a1b-GDF7; see, e.g., SEQ ID NO:39). In yet another embodiment, thedisclosure provides a chimeric TGF-beta polypeptide comprising a segmentfrom BMP-2 and segments from GDF-8 (e.g., a 1b-GDF8; see, e.g., SEQ IDNO:41). The chimeric polypeptides of the disclosure retain a TGF-betaprotein family member activity. Such activity can be measured in anynumber of ways as described below. In some embodiments, the chimericpolypeptide has BMP-2 activity, but is not inhibited by Noggin.

In some embodiments, segment of a chimeric polypeptide is 100% identicalto the parental strand from which the segment was derived. In otherembodiments the segment can comprise an amino acid sequence that has atleast 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity toa corresponding segment in a parental strand. For example, the segmentmay have one or more conservative amino acid substitutions (e.g., 1-5conservative amino acid substitutions).

In some embodiments, the chimeric TGF-beta family polypeptide may haveimproved activity compared to one or more of the parental strands fromwhich the chimeric polypeptide is generated. Biological activity of achimeric polypeptide of the disclosure can be measured using any numberof recognized assays in the art for TGF-beta activity. Such assaysinclude, but are not limited to, BIAcore (Surface Plasmon Resonance);C₂Cl₂ luciferase assay: Smad 1/5 reporter system; HEK293 luciferaseassay: Smad 2/3 reporter system; FSH (Follicle Stimulating Hormone)release assay: in rat pituitary cells; BRE (BMP Response Element)luciferase assay: Smad 1/5 reporter HEK 293 cells; Cripto binding assay:Luciferase response measured in presence/absence of Cripto; Human StemCell assay: Maintenance or Differentiation of H9 cells; NMR bindingStudies; Micro mass culture: Bone formation measured in Chick embryos;X-ray Crystallography: Determine Structure of ligand:receptor complexes;Native Gel: Visualization of ligand:receptor complexes; Size ExclusionChromatography (SEC): Visualization of ligand:receptor complexes;Velocity Scan Ultracentrifugation: Visualize ligand:receptor complexformation; and Seldi mass Spectrometry: Accurately determine size ofligands.

The chimeric TGF-beta family polypeptides described herein may beprepared in various forms, such as lysates, crude extracts, or isolatedpreparations.

In some embodiments, the isolated chimeric polypeptide is asubstantially pure polypeptide composition. A “substantially purepolypeptide” refers to a composition in which the polypeptide species isthe predominant species present (i.e., on a molar or weight basis it ismore abundant than any other individual macromolecular species in thecomposition), and is generally a substantially purified composition whenthe object species comprises at least about 50 percent of themacromolecular species present by mole or % weight. Generally, asubstantially pure polypeptide composition will comprise about 60% ormore, about 70% or more, about 80% or more, about 90% or more, about 95%or more, and about 98% or more of all macromolecular species by mole or% weight present in the composition. In some embodiments, the objectspecies is purified to essential homogeneity (i.e., contaminant speciescannot be detected in the composition by conventional detection methods)wherein the composition consists essentially of a single macromolecularspecies. Solvent species, small molecules (<500 Daltons), and elementalion species are not considered macromolecular species.

In certain embodiments, the disclosure contemplates making functionalvariants by modifying the structure of chimeras. Such modifications maybe made, for example, for such purposes as enhancing therapeuticefficacy, or stability (e.g., ex vivo shelf life and resistance toproteolytic degradation in vivo, improve stability, solubility,bioavailability, or biodistribution of the chimeric protein, etc.). Forexample, but not by way of limitation, the derivatives include chimerasthat have been modified, e.g., by acetylation, carboxylation, acylationglycosylation, pegylation, phosphorylation, farnesylation,biotinylation, lipidation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein such as an organic derivatizing agent, etc. Anyof numerous chemical modifications may be carried out by knowntechniques, including, but not limited to specific chemical cleavage,acetylation, formylation, metabolic synthesis, etc. Additionally, thederivative may contain one or more non-natural amino acids, such asthose with ketone-containing side chain, polyethylene glycols, lipids,poly- or mono-saccharide, and phosphates. Effects of such non-naturalamino acid elements on the functionality of a chimeric TGF-betasuperfamily protein may be tested as described herein for other TGF-betasuperfamily protein variants. When a chimeric TGF-beta superfamilyprotein is produced in cells by cleaving a nascent form of the precursorprotein, post-translational processing may also be important for correctfolding and/or function of the protein. Different cells (such as CHO,HeLa, MDCK, 293, W138, NIH-3T3 or HEK293) have specific cellularmachinery and characteristic mechanisms for such post-translationalactivities and may be chosen to ensure the correct post-translationalmodification and processing of the precursor protein into a chimericTGF-beta superfamily protein. In vitro cell-free expression system incombination with its associated engineered tRNA synthase and tRNA can beutilized to ensure the correct modification in a specific amino acidposition genetically tagged to introduce non-natural amino acids.

Modified chimeras can also be produced, for instance, by amino acidsubstitution, deletion, or addition. For instance, it is reasonable toexpect that an isolated replacement of a leucine with an isoleucine orvaline, an aspartate with a glutamate, a threonine with a serine, or asimilar replacement of an amino acid with a structurally related aminoacid (e.g., conservative mutations) will not have a major effect on thebiological activity of the resulting molecule. Conservative replacementsare those that take place within a family of amino acids that arerelated in their side chains.

In certain embodiments, the disclosure contemplates making mutations ina proteolytic cleavage site of the chimera sequence to make the siteless susceptible to proteolytic cleavage. Computer analysis (using acommercially available software, e.g., MacVector, Omega, PCGene,Molecular Simulation, Inc.) can be used to identify proteolytic cleavagesites. As will be recognized by one of skill in the art, most of thedescribed mutations, variants or modifications may be made at thenucleic acid level or, in some cases, by post translational modificationor chemical synthesis. Such techniques are well known in the art.

In certain embodiments, the disclosure contemplates specific mutationsof a chimera sequences so as to alter the glycosylation of the chimera.Such mutations may be selected so as to introduce or eliminate one ormore glycosylation sites, such as O-linked or N-linked glycosylationsites. Asparagine-linked glycosylation recognition sites generallycomprise a tripeptide sequence, asparagine-X-threonine (where “X” is anyamino acid) which are specifically recognized by appropriate cellularglycosylation enzymes. The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the wild-type polypeptide (for O-linked glycosylationsites). A variety of amino acid substitutions or deletions at one orboth of the first or third amino acid positions of a glycosylationrecognition site (and/or amino acid deletion at the second position)results in non-glycosylation at the modified tripeptide sequence.Another means of increasing the number of carbohydrate moieties is bychemical or enzymatic coupling of glycosides to the polypeptide.Depending on the coupling mode used, the sugar(s) may be attached to (a)arginine and histidine; (b) free carboxyl groups; (c) free sulfhydrylgroups such as those of cysteine; (d) free hydroxyl groups such as thoseof serine, threonine, or hydroxyproline; (e) aromatic residues such asthose of phenylalanine, tyrosine, or tryptophan; or (f) the amide groupof glutamine. These methods are described in WO 87/05330 published Sep.11, 1987, and in Aplin and Wriston (1981) CRC Crit. Rev. Biochem., pp.259-306, incorporated by reference herein. Removal of one or morecarbohydrate moieties present on a chimera may be accomplishedchemically and/or enzymatically. Chemical deglycosylation may involve,for example, exposure to the compound trifluoromethanesulfonic acid, oran equivalent compound. This treatment results in the cleavage of mostor all sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the amino acid sequence intact.Chemical deglycosylation is further described by Hakimuddin et al.(1987) Arch. Biochem. Biophys. 259:52 and by Edge et al. (1981) Anal.Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al. (1987) Meth. Enzymol.138:350. The nucleic acid and/or amino acid sequence of a propeptide maybe adjusted, as appropriate, depending on the type of expression systemused, as mammalian, yeast, insect and plant cells may all introducediffering glycosylation patterns that can be affected by the amino acidsequence of the peptide.

In some embodiments, the chimeric polypeptide can be in the form ofarrays. The polypeptide may be in a soluble form, for example assolutions in the wells of mircotitre plates, or immobilized onto asubstrate. The substrate can be a solid substrate or a porous substrate(e.g, membrane), which can be composed of organic polymers such aspolystyrene, polyethylene, polypropylene, polyfluoroethylene,polyethyleneoxy, and polyacrylamide, as well as co-polymers and graftsthereof. A solid support can also be inorganic, such as glass, silica,controlled pore glass (CPG), reverse phase silica or metal, such as goldor platinum. The configuration of a substrate can be in the form ofbeads, spheres, particles, granules, a gel, a membrane or a surface.Surfaces can be planar, substantially planar, or non-planar. Solidsupports can be porous or non-porous, and can have swelling ornon-swelling characteristics. A solid support can be configured in theform of a well, depression, or other container, vessel, feature, orlocation. A plurality of supports can be configured on an array atvarious locations, addressable for robotic delivery of reagents, or bydetection methods and/or instruments.

The disclosure also provides polynucleotides encoding the chimericTGF-beta family polypeptides disclosed herein. The polynucleotides maybe operably linked to one or more heterologous regulatory or controlsequences that control gene expression to create a recombinantpolynucleotide capable of expressing the polypeptide. Expressionconstructs containing a polynucleotide encoding the chimeric polypeptidecan be introduced into appropriate host cells to express thepolypeptide. Polynucleotide sequences encoding various domains or fullchimera of the disclosure can be determined without undue efforts basedupon the various codons that are associated with an amino acid of in apolypeptide. Furthermore, the disclosure provides exemplary sequences ofthe TGF-β family member. Deriving the sequences of a domain or chimerafrom the sequences provided herein is readily performed by one of skillin the art. Given the knowledge of specific sequences of the TGF-betafamily of proteins, and the specific descriptions of the chimericpolypeptides herein (e.g., the segment structure of the chimericdomains), the nucleic acid sequence of the engineered chimera will beapparent to the skilled artisan. The knowledge of the codonscorresponding to various amino acids coupled with the knowledge of theamino acid sequence of the polypeptides allows those skilled in the artto make different polynucleotides encoding the polypeptides of thedisclosure. Thus, the present disclosure contemplates each and everypossible variation of the polynucleotides that could be made byselecting combinations based on possible codon choices, and all suchvariations are to be considered specifically disclosed for any of thepolypeptides described herein.

In some embodiments, the polynucleotides comprise polynucleotides thatencode the polypeptides described herein but have about 80% or moresequence identity, about 85% or more sequence identity, about 90% ormore sequence identity, about 91% or more sequence identity, about 92%or more sequence identity, about 93% or more sequence identity, about94% or more sequence identity, about 95% or more sequence identity,about 96% or more sequence identity, about 97% or more sequenceidentity, about 98% or more sequence identity, or about 99% or moresequence identity at the nucleotide level to a reference polynucleotideencoding a chimera or parental TGF-beta family polypeptide.

In some embodiments, the isolated polynucleotides encoding thepolypeptides may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the isolatedpolynucleotide prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotides and nucleic acid sequences utilizingrecombinant DNA methods are well known in the art. Guidance is providedin Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rdEd., Cold Spring Harbor Laboratory Press; and Current Protocols inMolecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998,updates to 2007.

In some embodiments, the polynucleotides are operatively linked tocontrol sequences for the expression of the polynucleotides and/orpolypeptides. In some embodiments, the control sequence may be anappropriate promoter sequence, which can be obtained from genes encodingextracellular or intracellular polypeptides, either homologous orheterologous to the host cell.

In some embodiments, the control sequence may also be a suitabletranscription terminator sequence, a sequence recognized by a host cellto terminate transcription. The terminator sequence is operably linkedto the 3′ terminus of the nucleic acid sequence encoding thepolypeptide. Any terminator which is functional in the host cell ofchoice may be used.

In some embodiments, the control sequence may also be a suitable leadersequence, a nontranslated region of an mRNA that is important fortranslation by the host cell. The leader sequence is operably linked tothe 5′ terminus of the nucleic acid sequence encoding the polypeptide.Any leader sequence that is functional in the host cell of choice may beused.

In some embodiments, the control sequence may also be a signal peptidecoding region that codes for an amino acid sequence linked to the aminoterminus of a polypeptide and directs the encoded polypeptide into thecell's secretory pathway. The 5′ end of the coding sequence of thenucleic acid sequence may inherently contain a signal peptide codingregion naturally linked in translation reading frame with the segment ofthe coding region that encodes the secreted polypeptide. Alternatively,the 5′ end of the coding sequence may contain a signal peptide codingregion that is foreign to the coding sequence. The foreign signalpeptide-coding region may be required where the coding sequence does notnaturally contain a signal peptide coding region.

The disclosure is further directed to a recombinant expression vectorcomprising a polynucleotide encoding the chimeric TGF-beta polypeptidesdescribed herein, and one or more expression regulating regions such asa promoter and a terminator, a replication origin, etc., depending onthe type of hosts into which they are to be introduced. In creating theexpression vector, the coding sequence is located in the vector so thatthe coding sequence is operably linked with the appropriate controlsequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the polynucleotidesequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell or in vitro cell-freereaction mixture into which the vector is to be introduced. The vectorsmay be linear or closed circular plasmids.

The expression vector may be an autonomously replicating vector, i.e., avector that exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon, may be used.

In some embodiments, the expression vector of the disclosure containsone or more selectable markers, which permit easy selection oftransformed cells. A selectable marker is a gene the product of whichprovides for biocide or viral resistance, resistance to heavy metals,prototrophy to auxotrophs, and the like.

In another embodiments, the disclosure provides a host cell comprising apolynucleotide encoding the chimeric TGF-beta polypeptide, thepolynucleotide being operatively linked to one or more control sequencesfor expression of the fusion polypeptide in the host cell. Host cellsfor use in expressing the fusion polypeptides encoded by the expressionvectors of the present disclosure are well known in the art. Appropriateculture mediums and growth conditions for the above-described host cellsare well known in the art.

Expression vectors can be designed for expression of chimeras inprokaryotic or eukaryotic cells. For example, chimeras of the disclosurecan be expressed in bacterial or prokaryote cells such as E. Coli,insect cells (e.g., the baculovirus expression system), yeast cells,microalgae, plant cells or mammalian cells as well as in vitro cell-freeexpression system. Some suitable host cells are discussed further inGoeddel, Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990).

While one example of an expression system discussed is an E. coliexpression system, to those skilled in the art, these proteins can beeasily be cloned into and expressed from a large number of otherexpression systems. The advantages include, but are not limited to,achieving post-translational modifications as would be seen in theorganism the protein was derived from (in this case H. sapiens),expression of the ligands without the start methionine required forbacterial expression, and easy incorporation of non-natural amino acidsor additional chemical modifications. Suitable prokaryotes include butare not limited to eubacteria, such as Gram-negative or Gram-positiveorganisms, for example, Enterobacteriaceae such as E. coli. Various E.coli strains are publicly available, such as E. coli K12 strain MM294(ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC27,325) and K5 772 (ATCC 53,635). In addition to prokaryotes, eukaryoticmicrobes such as filamentous fungi or yeast are suitable cloning orexpression hosts for VEGF-E-encoding vectors. Saccharomyces cerevisiaeis a commonly used lower eukaryotic host microorganism.

Suitable host cells for the expression of chimeras are derived fromunicellular and multicellular organisms. Examples of invertebrate cellsinclude insect cells such as Drosophila S2 and Spodoptera Sf9, as wellas plant cells. Plant expression systems have also been usedsuccessfully to express modified proteins. Examples of useful mammalianhost cell lines include Chinese hamster ovary (CHO) and COS cells. Morespecific examples include monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. GenVirol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR(CHO, Urlaub andChasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells(TM4, Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammarytumor (MMT 060562, ATCC CCL51). The selection of the appropriate hostcell is deemed to be within the skill in the art.

Alternate protein expression systems include human embryonic kidney(HEK) 293 cells, insect cell line (S. frugiperda) utilizing thebaculovirus expression system, yeast expression systems not limited toP. pastoris and S. cerevisiae, and numerous Microalgae strains.Transgenic animals can be used to express correctly modified protein. Inessence, the animals become living ‘bioreactors’ capable of expressinglarge amounts of the desired protein in an easily harvested fluid ortissue, such as the milk from a cow. Cell-free in vitro expressionsystems using either the bacterial or wheat germ cell lysate can beemployed. Cell-free expression system will permit inserting a wide rangeof non-natural amino acids or epitope tags with higher efficiency andgreater specificity.

Examples of bacterial vectors include pQE70, pQE60, pQE-9 (Qiagen), pBS,pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, andpRIT5 (Pharmacia). Examples of vectors for expression in the yeast S.cerevisiae include pYepSec1 (Baldari et al., EMBO J. 6:229 (1987)), pMFa(Kurjan and Herskowitz, Cell 30:933 (1982)), pJRY88 (Schultz et al.,Gene 54:113 (1987)), and pYES2 (Invitrogen Corporation, San Diego,Calif.). Baculovirus vectors available for expression of nucleic acidsto produce proteins in cultured insect cells (e.g., Sf9 cells) includethe pAc series (Smith et al., Mol. Cell. Biol. 3:2156 (1983)) and thepVL series (Lucklow and Summers Virology 170:31 (1989)).

Examples of mammalian expression vectors include pWLNEO, pSV2CAT, p0G44,pXT1, pSG (Stratagene) pSVK3, PBPV, pMSG, PSVL (Pharmacia), pCDM8 (Seed,Nature 329:840 (1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187(1987)). When used in mammalian cells, the expression vector's controlfunctions are often provided by viral regulatory elements. For example,commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus and Simian Virus 40.

Viral vectors have been used in a wide variety of gene deliveryapplications in cells, as well as living animal subjects. Viral vectorsthat can be used include, but are not limited to, retrovirus,lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus,vaccinia virus, herpes virus, Epstein-Barr virus, adenovirus,geminivirus, and caulimovirus vectors. Non-viral vectors includeplasmids, liposomes, electrically charged lipids (cytofectins), nucleicacid-protein complexes, and biopolymers. In addition to a nucleic acidof interest, a vector may also comprise one or more regulatory regions,and/or selectable markers useful in selecting, measuring, and monitoringnucleic acid transfer results (delivery to specific tissues, duration ofexpression, etc.).

The chimera of the disclosure can be made by using methods well known inthe art. Polynucleotides can be synthesized by recombinant techniques,such as that provided in Sambrook et al., 2001, Molecular Cloning: ALaboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press; andCurrent Protocols in Molecular Biology, Ausubel. F. ed., Greene Pub.Associates, 1998, updates to 2007. Polynucleotides encoding the enzymes,or the primers for amplification can also be prepared by standardsolid-phase methods, according to known synthetic methods, for exampleusing phosphoramidite method described by Beaucage et al., (1981) TetLett 22:1859-69, or the method described by Matthes et al., (1984) EMBOJ. 3:801-05, e.g., as it is typically practiced in automated syntheticmethods. In addition, automated peptide synthesizers are commerciallyavailable (e.g., Advanced ChemTech Model 396; Milligen/Biosearch 9600).

In a one embodiment, the disclosure is directed to a method toaccelerate construction of large chimera libraries. Accordingly, thedisclosure provides a recombinant strategy termed RASCH (RAndomSegmental CHimera) See FIG. 17. It uses a template sequence (firststrand from one TGF-beta superfamily member) and a few target sequences(second (third, fourth, fifth, sixth) strand from one or more alternateTGF-beta superfamily members), whose subdomains are to be linked. Thetemplate DNA sequence is used to promote efficient coupling of thetarget sequences and is degraded once subdomains are linked. Followingthe gene construction to create the chimeric sequences, the new ligandsare chemically refolded into functional dimer. This dimerization processpermits additional diversification of the final sequence by mixing anddimerizing two different sequences of both natural and designer origins.Therefore, the RASCH method can be used to diversify the approximate 40natural protein sequences of TGF-beta superfamily ligands into ten ofthousands or more variant sequences, each distinct from anynaturally-occurring TGF-beta superfamily ligand sequences.

Engineered polypeptide expressed in a host cell can be recovered fromthe cells and or the culture medium using any one or more of the wellknown techniques for protein purification, including, among others,lysozyme treatment, sonication, filtration, salting-out,ultra-centrifugation, chromatography, and affinity separation (e.g.,substrate bound antibodies).

Chromatographic techniques for isolation of the polypeptides include,among others, reversed phase chromatography high performance liquidchromatography, ion exchange chromatography, gel electrophoresis, andaffinity chromatography. Conditions for purifying a particular enzymewill depend, in part, on factors such as net charge, hydrophobicity,hydrophilicity, molecular weight, molecular shape, etc., and will beapparent to those having skill in the art.

Assays to determine activity are well known in the art. The presentdisclosure relates to assays to test for biological activity of chimericproteins, more preferably, to assays to test for clinical activity. Suchactivity can include enhanced agonistic or antagonistic TGF-betaactivity, combined or novel biological activity, and the like.

In certain embodiments, a chimeric protein of the disclosure comprisingan agonist of a TGF-beta superfamily protein comprises an antagonist ofa different TGF-beta superfamily protein.

Irrespective of which protein expression, harvesting, and, foldingmethodologies are used, certain of the subject chimeric proteins canbind, preferentially to a pre-selected receptor and can now beidentified using standard methodologies, e.g., ligand/receptor bindingassays, well known, and thoroughly documented in the art. See, e.g.,Legerski gl al. (1992) Bio h⁻_Biophys. Res. Comm. 183: 672679; Frakar etal. (1978) Biochem. Bio12-hys. Res. Comm 80:849-857; Chio et el. (1990)Nature 343: 266-269; Dahlman et al. (1988) Biochem 27: 1813-1817;Strader et el. (1989) J. Biol. Chem. 264: 13572-13578; and DDowd et al.(1988) J. Biol. Chem. 263: 15985-15992.

Typically, in a ligand/receptor binding assay, the native or parentTGF-beta superfamily member of interest having a known, quantifiableaffinity for a pre-selected receptor is labeled with a detectablemoiety, for example, a radiolabel, a chromogenic label, or a fluorogeniclabel. Aliquots of purified receptor, receptor binding domain fragments,or cells expressing the receptor of interest on their surface areincubated with the labeled TGF-beta superfamily member in the presenceof various concentrations of the unlabeled chimeric protein. Therelative binding affinity of a candidate chimeric protein may bemeasured by quantitating the ability of the chimeric protein to inhibitthe binding of the labeled TGF-beta superfamily member with thereceptor. In performing the assay, fixed concentrations of the receptorand the TGF-beta superfamily member are incubated in the presence andabsence of unlabeled chimeric protein. Sensitivity may be increased bypreincubating the receptor with the chimeric protein before adding thelabeled template TGF-beta superfamily member. After the labeledcompetitor has been added, sufficient time is allowed for adequatecompetitor binding, and then free and bound labeled TGF-beta superfamilymembers are separated from one another, and one or the other measured.Labels useful in the practice of the screening procedures includeradioactive labels, chromogenic labels, spectroscopic labels such asthose disclosed in Haughland (1994) “Handbook of Fluorescent andResearch Chemicals,” 5 ed. by Molecular Probes, Inc., Eugene, Oreg., orconjugated enzymes having high turnover rates, i.e., horseradishperoxidase, alkaline phosphatase, or agalactosidase, used in combinationwith chemiluminescent or fluorogenic substrates. The biologicalactivity, namely the agonist or antagonist properties of the resultingchimeric protein constructs can subsequently be characterized usingconventional in vivo and in vitro assays that have been developed tomeasure the biological activity of any TGF-beta superfamily member. Itis appreciated, however, the type of assay used preferably depends onthe TGF-a superfamily member upon which the chimeric protein is based.For example, chimeric constructs based upon naturally occurring BMP-2protein may be assayed using any of the biological assays that have beendeveloped to date for measuring BMP-2 activity, described in more detailbelow.

The presence of multimers among the subject chimeric proteins can bedetected visually either by standard SDS-PAGE in the absence of areducing agent such as DTT or by HPLC (e.g., C18 reverse phase HPLC).Multimeric proteins of the present disclosure can have an apparentmolecular weight proportionally greater than the monomeric subunit,e.g., in the range about 28-36 kDa for a dimer, as compared to monomericsubunits, which may have an apparent molecular weight of about 14-18kDa. The multimeric protein can readily be visualized on anelectrophoresis gel by comparison to commercially available molecularweight standards. The dimeric protein also elutes from a C18 RP HPLC(45-50% acetonitrile: 0.1% TFA) at a time point different from that forits monomeric counterpart.

A second assay evaluates the presence of dimer (e.g., OP-1 dimers) byits ability to bind to hydroxyapatite. Optimally-folded dimer binds ahydroxyapatite column well in pH7, 10 mM phosphate, 6M urea, and 0.142MNaCl (dimer elutes at 0.25 M NaCl) as compared to monomer, which doesnot bind substantially at those concentrations (monomer elutes at 0.1MNaCl). A third assay evaluates the presence of dimer by the protein'sresistant to trypsin or pepsin digestion. The folded dimeric species issubstantially resistant to both enzymes, particularly trypsin, whichcleaves only a small portion of the N-terminus of the mature protein,leaving a biologically active dimeric species only slightly smaller insize than the untreated dimer (each monomer in amino acids smaller aftertrypsin cleavage). By contrast, the monomers and misfolded dimers aresubstantially degraded. In the assay, the protein is subjected to anenzyme digest using standard conditions, e.g., digestion in a standardbuffer such as 50 mM Tris buffer, pH 8, containing 4 M urea, 100 mMNaCl, 0.3% Tween-80 and 20 mM methylamine. Digestion is allowed to occurat 37° C. for on the order of 16 hours, and the product visualized byany suitable means, preferably SDS PAGE.

The biological activity of the subject chimeric proteins, for example,the chimeric proteins having one or more segments from BMPs, can beassessed by any of a number of means as described in WO00/20607. Forexample, the protein's ability to induce endochondral bone formation canbe evaluated using the well characterized rat subcutaneous bone assay.In the assay bone formation is measured by histology, as well as byalkaline phosphatase and/or osteoclacin production. In addition,osteogenic proteins having high specific bone forming activity, such asOP-1, BMP-2, BMR4, BMP-5 and BMP-6, also induce alkaline phosphataseactivity in an in vitro rat osteoblast or osteosarcoma cell-based assay.Such assays are well described in the art. See, for example, Sabokdar ofal. (1994) Bone and Mineral 27:57-67.; Knutsen et al. (1993) BiochemBiophys Res. Commun 194:1352-1358; and Maliakal et al. (1994) GrowthFactors 1:227-234).

By contrast, osteogenic proteins having low specific bone formingactivity, such as CDMP-1 and CDMP-2, for example, do not induce alkalinephosphatase activity in the cell based osteoblast assay. The assay thusprovides a ready method for evaluating biological activity of B1b9mutants. For example, CDMP-1, CDMP-2 and CMDP-3 all are competent toinduce bone formation, although with a lower specific activity thanBMP-2, BW-4, BV-5, BMP-6 or OP-1. Conversely, BMP-2, BMP-4, BMP-5,BPy1P-6 and OP-1 all can induce articular cartilage formation, albeitwith a lower specific activity than CDMP-1, CDMP-2 or CDMP-3.Accordingly, a chimeric protein having one or more segment from CDMP,designed and described herein to be a chimeric protein competent toinduce alkaline phosphatase activity in the cell-based assay, isexpected to demonstrate a higher specific bone forming activity in therat animal bioassay.

The chimeric protein's biological activity can also be readily evaluatedby the protein's ability to inhibit epithelial cell growth. A useful,well characterized in vitro assay utilizes mink lung cells or melanomacells. See WO00/20607. Other assays for other members of the TGF-betasuperfamily are well described in the literature and can be performedwithout undue experimentation.

In certain embodiment, the disclosure provides methods and agents forcontrol and maintain skeletal muscle mass in a host, preferably a human.Therefore, any chimeric protein of the disclosure that is expected toaffect muscle-related function of a TGF-beta superfamily protein such asfor example GDF-8 can be tested in whole cells or tissues, in vitro orin vivo, to confirm their ability to modulate skeletal muscle mass.GDF-8 (also known as myostatin) is a negative regulator of skeletalmuscle growth. GDF-8 knockout mice have approximately twice the skeletalmuscle mass of normal mice. The effects of increased muscle mass on bonemodeling may be investigated, e.g., by examining bone mineral content(BMC) and bone mineral density (BMD) in the femora of female GDF-8knockout mice. Dual-energy X-ray absorptiometry (DEXA) densitometry canbe used to measure whole-femur BMC and BMD, and PQCT densitometry can beused to calculate BMC and BMD from cross-sections of tissues. Hamrick,Anat Rec. 2003 May; 272A(1):388-91. As is known in the art, a chimericprotein of the disclosure may be introduced into the GDF-8 knockoutmice, and similar assays can be used to determine the effect of thechimeric protein on skeletal muscle mass and bone density.

The dystrophic phenotype in the mdx mouse model of Duchenne musculardystrophy (DMD) may also be employed to test the biological activity ofa chimeric protein of the disclosure. It was reported that blockade ofendogenous myostatin by using intraperitoneal injections of blockingantibodies for three months resulted in an increase in body weight,muscle mass, muscle size and absolute muscle strength in mdx mousemuscle along with a significant decrease in muscle degeneration andconcentrations of serum creatine kinase. Bogdanovich et al., Nature.2002 Nov. 28; 420(6914):418-21. Similar study may be employed todetermine whether a chimeric protein of the disclosure potentiates orinhibits the endogenous GDF-8 activity.

In certain embodiments, the disclosure provides methods and agents formodulating neurogenesis. For example, GDF-11 is known to inhibitolfactory epithelium neurogenesis in vitro by inducing p27(Kip1) andreversible cell cycle arrest in progenitors. Wu et al. Neuron. 2003 Jan.23; 37(2):197-207. The effect of a chimeric protein of the disclosure onneurogenesis can be similarly tested. Further, the effect of a chimericprotein of the disclosure on GDF-11's effect on neurogenesis can also betested using similar assays as described in Wu et al. Id.

In certain embodiment, the disclosure provides methods and agents forstimulating bone formation and increasing bone mass. Therefore, anychimeric protein of the disclosure that is expected to affectbone-related function of a TGF-beta superfamily protein such as forexample BMP-2, BMP-3, GDF-10, BMP-4, BMP-7, or BMP-8, can be tested inwhole cells or tissues, in vitro or in vivo, to confirm their ability tomodulate bone or cartilage growth. Various methods known in the art canbe utilized for this purpose. For example, BMP-3 inhibits BMP2-mediatedinduction of Msx2 and blocks BMP2-mediated differentiation ofosteoprogenitor cells into osteoblasts. Thus, the effect of a subjectchimer protein, preferably one comprising a segment from a BMP-2 orBMP-3, on bone or cartilage growth can be determined by their effect onthe osteogenic activity of BMP-2, for example, by measuring induction ofMsx2 or differentiation of osteoprogenitor cells into osteoblasts incell based assays (see, e.g., Daluiski et al., Nat. Genet. 2001,27(1):84-8; Hino et al., Front Biosci. 2004, 9:1520-9). Similarly, asubject chimeric protein, preferably one comprising a segment from aBMP-2 or BMP-3, may be tested for its osteogenic or anti-osteogenicactivity or its agonistic or antagonistic effect on BMP-2-mediatedosteogenesis.

Another example of cell-based assays includes analyzing the osteogenicor anti-osteogenic activity of a subject chimeric and test compounds inmesenchymal progenitor and osteoblastic cells. To illustrate,recombinant adenoviruses expressing a subject chimeric protein wereconstructed to infect pluripotent mesenchyimal progenitor C3H10T1/2cells, preosteoblastic C2C12 cells, and osteoblastic TE-85 cells.Osteogenic activity is then determined by measuring the induction ofalkaline phosphatase, osteocalcin, and matrix mineralization (see, e.g.,Cheng et al., J bone Joint Surg Am. 2003, 85-A(8):1544-52).

Further, the disclosure contemplates in vivo assays to measure bone orcartilage growth. For example, Namkung-Matthai et al., Bone, 28:80-86(2001) discloses a rat osteoporotic model in which bone repair duringthe early period after fracture is studied. Kubo et al., SteroidBiochemistry & Molecular Biology, 68:197-202 (1999) also discloses a ratosteoporotic model in which bone repair during the late period afterfracture is studied. These references are incorporated by referenceherein in their entirety for their disclosure of rat model for study onosteoporotic bone fracture. In certain aspects, the present disclosuremakes use of fracture healing assays that are known in the art. Theseassays include fracture technique, histological analysis, andbiomechanical analysis, which are described in, for example, U.S. Pat.No. 6,521,750, which is incorporated by reference in its entirety forits disclosure of experimental protocols for causing as well asmeasuring the extent of fractures, and the repair process.

It is understood that the screening assays of the disclosure apply tonot only the subject chimeric proteins and variants thereof, but alsoany test compounds including agonists and antagonist of the chimericproteins or their variants themselves. Further, these screening assaysare useful for drug target verification and quality control purposes.

In other embodiment, the disclosure relates to the use of the subjectchimeric TGF-beta superfamily proteins to identify compounds which canmodulate activities of the chimeric proteins. Compounds identifiedthrough this screening can be tested in tissues (e.g., bone and/orcartilage) or cells (e.g., muscle cells) to assess their ability tomodulate the test tissues or cells (e.g., bone/cartilage growth ormuscle cell growth) in vitro. Optionally, these compounds can further betested in animal models to assess their ability to modulate, e.g.,bone/cartilage growth or muscle control and maintenance in vivo.

A variety of assay formats will suffice and, in light of the disclosure,those not expressly described herein will nevertheless be comprehendedby one of ordinary skill in the art. As described herein, the testcompounds (agents) of the disclosure may be created by any combinatorialchemical method. Alternatively, the subject compounds may be naturallyoccurring biomolecules synthesized in vivo or in vitro. Compounds(agents) to be tested for their ability to act as modulators of bone orcartilage growth can be produced, for example, by bacteria, yeast,plants or other organisms (e.g., natural products), produced chemically(e.g., small molecules, including peptidomimetics), or producedrecombinantly. Test compounds contemplated by the present disclosureinclude non-peptidyl organic molecules, peptides, polypeptides,peptidomimetics, sugars, hormones, and nucleic acid molecules. In aspecific embodiment, the test agent is a small organic molecule having amolecular weight of less than about 2,000 daltons.

The test compounds of the disclosure can be provided as single, discreteentities, or provided in libraries of greater complexity, such as madeby combinatorial chemistry. These libraries can comprise, for example,alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers andother classes of organic compounds. Presentation of test compounds tothe test system can be in either an isolated form or as mixtures ofcompounds, especially in initial screening steps. Optionally, thecompounds may be optionally derivatized with other compounds and havederivatizing groups that facilitate isolation of the compounds.Non-limiting examples of derivatizing groups include biotin,fluorescein, digoxygenin, green fluorescent protein, isotopes,polyhistidine, magnetic beads, glutathione S transferase,photoactivatible crosslinkers or any combinations thereof.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity or bioavailability of the test compound canbe generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity between a chimericTGF-beta superfamily protein and its binding protein (e.g., the chimericprotein itself or a TGF-beta receptor protein or fragments thereof).

Merely to illustrate, in an exemplary screening assay of the presentdisclosure, the compound of interest is contacted with an isolated andpurified chimeric protein which is ordinarily capable of binding to aTGF-beta receptor protein or fragments thereof, as appropriate for theintention of the assay. To the mixture comprising a subject chimericprotein and a TGF-beta receptor protein is then added a compositioncontaining a test compound. Detection and quantification of the chimericprotein receptor complexes provides a means for determining thecompound's efficacy at inhibiting (or potentiating) complex formationbetween the chimeric TGF-beta superfamily protein and its bindingprotein, e.g., the TGF-beta receptor or fragments thereof. The efficacyof the compound can be assessed by generating dose response curves fromdata obtained using various concentrations of the test compound.Moreover, a control assay can also be performed to provide a baselinefor comparison. For example, in a control assay, an isolated andpurified chimeric TGF-beta superfamily protein is added to a composition(cell-free or cell-based) containing a TGF-beta receptor protein orfragment thereof, and the formation of the chimeric protein-receptorcomplex is quantitated in the absence of the test compound. It will beunderstood that, in general, the order in which the reactants may beadmixed can be varied, and can be admixed simultaneously. Moreover, inplace of purified proteins, cellular extracts and lysates may be used torender a suitable cell-free assay system. Alternatively, cellsexpressing a TGF-beta receptor protein or fragments thereof on theirsurfaces can be used in certain assays.

Complex formation between a subject chimeric TGF-beta superfamilyprotein and its binding protein may be detected by a variety oftechniques. For instance, modulation of the formation of complexes canbe quantitated using, for example, detectably labeled proteins such asradiolabelled (e.g., 32 P, 35 S, 14 C or 3H), fluorescently labeled(e.g., FITC), or enzymatically labeled chimeric protein or its bindingprotein, by immunoassay, or by chromatographic detection.

In certain embodiments, the present disclosure contemplates the use offluorescence polarization assays and fluorescence resonance energytransfer (FRET) assays in measuring, either directly or indirectly, thedegree of interaction between a chimeric TGF-beta superfamily proteinand its binding protein (e.g., a TGF-beta receptor protein or fragmentsthereof). Further, other modes of detection such as those based onoptical waveguides (PCT Publication WO 96/26432 and U.S. Pat. No.5,677,196), surface plasmon resonance (SPR), surface charge sensors, andsurface force sensors are compatible with many embodiments of thedisclosure.

Moreover, the present disclosure contemplates the use of an interactiontrap assay, also known as the “two hybrid assay,” for identifying agentsthat disrupt or potentiate interaction between a chimeric TGF-betasuperfamily protein and its binding protein (e.g., a TGF-beta receptorprotein or fragments thereof). See for example, U.S. Pat. No. 5,283,317;Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; andIwabuchi et al. (1993) Oncogene 8:1693-1696).

Chimera polynucleotides, polypeptides, antibodies, cells and otherreagents of the disclosure have a wide variety of uses, both in vitroand in vivo. For example, in representative embodiments, these reagentsmay be used in vitro or in vivo (e.g., in an animal model) to study theprocesses of mineralization, bone formation, and bone loss. Further,“knock in” and “knock out” animals can be used as animal models ofdisease or as screening tools (discussed more below) for compounds thatinteract with the chimera polynucleotides or polypeptides. It will beapparent to those skilled in the art that any suitable vector can beused to deliver the polynucleotide to a cell or subject. The choice ofdelivery vector can be made based on a number of factors known in theart, including age and species of the target host, in vitro versus invivo delivery, level and persistence of expression desired, intendedpurpose (e.g., for therapy or screening), the target cell or organ,route of delivery, size of the isolated polynucleotide, safety concerns,and the like.

Chimeric polypeptide of the disclosure may be formulated for use invarious biological systems including in vivo. Any of a variety ofart-known methods can be used to administer a chimera either alone or incombination with other active agents. For example, administration can beparenterally by injection or by gradual infusion over time. The agent(s)can be administered by such means as oral, rectal, buccal (e.g.,sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscularincluding skeletal muscle, cardiac muscle, diaphragm muscle and smoothmuscle, intradermal, intravenous, intraperitoneal), topical (i.e., bothskin and mucosal surfaces, including airway surfaces), intranasal,transdermal, intraarticular, intrathecal, intracavity, and inhalationadministration, administration to the liver by intraportal delivery, aswell as direct organ injection (e.g., into the liver, into the brain fordelivery to the central nervous system, into the pancreas). The mostsuitable route in any given case will depend on the nature and severityof the condition being treated and on the nature of the particularcompound which is being used.

The disclosure also provides a pharmaceutical preparation comprising asubject chimeric protein and a pharmaceutically acceptable carrier. Apharmaceutical preparation may be employed to promote growth of a tissueor diminishing or prevent loss of a tissue in a subject, preferably ahuman. The targeted tissue can be, for example, bone, cartilage,skeletal muscle, cardiac muscle and/or neuronal tissue.

In another aspect, a chimeric TGF-beta polypeptide can be formulatedeither alone or in combination with other agents for administration(e.g., as a lotion, cream, spray, gel, or ointment). It may beformulated into liposomes to reduce toxicity or increasebioavailability. Other methods for delivery include oral methods thatentail encapsulation of the in microspheres or proteinoids, aerosoldelivery (e.g., to the lungs), or transdermal delivery (e.g., byiontophoresis or transdermal electroporation). Other methods ofadministration will be known to those skilled in the art.

Preparations for parenteral administration of a composition comprising achimeric TGF-beta polypeptide include sterile aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils (e.g., oliveoil), and injectable organic esters such as ethyl oleate. Examples ofaqueous carriers include water, saline, and buffered media,alcoholic/aqueous solutions, and emulsions or suspensions. Examples ofparenteral vehicles include sodium chloride solution, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's, and fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives such as, otherantimicrobial, anti-oxidants, cheating agents, inert gases and the likealso can be included.

The disclosure provides various disease and disorders that may bemodulated by a TGF-beta protein family member comprising contacting oradministering a therapeutically effective amount of a chimeric TGF-betapolypeptide either alone or in combination with other agents to asubject who has, or is at risk of having, such a disorder.

A therapeutically effective amount can be measured as the amountsufficient to decrease a subject's symptoms associated with the diseasesor disorder. Typically, the subject is treated with an amount of atherapeutic composition sufficient to reduce a symptom of a disease ordisorder by at least 50%, 90% or 100%. Generally, the optimal dosagewill depend upon the disorder and factors such as the weight of thesubject, the age, the weight, sex, and degree of symptoms. For example,with respect to bone morphogenesis, optionally, the dosage may vary withthe type of matrix used in the reconstitution and the types of compoundsin the composition. The addition of other known growth factors to thefinal composition, may also affect the dosage. Progress can be monitoredby periodic assessment of bone growth and/or repair, for example,X-rays, histomorphometric determinations, and tetracycline labeling.Nonetheless, suitable dosages can readily be determined by one skilledin the art. Typically, a suitable dosage is 0.5 to 40 mg/kg body weight,e.g., 1 to 8 mg/kg body weight.

As mentioned previously, the compositions and methods of the disclosurecan include the use of additional (e.g., in addition to a chimericTGF-beta polypeptide) therapeutic agents (e.g., an inhibitor of TNF, anantibiotic, and the like). The chimeric TGF-beta polypeptide, othertherapeutic agent(s), and/or antibiotic(s) can be administered,simultaneously, but may also be administered sequentially.

A pharmaceutical composition comprising a chimera according to thedisclosure can be in a form suitable for administration to a subjectusing carriers, excipients, and additives or auxiliaries. Frequentlyused carriers or auxiliaries include magnesium carbonate, titaniumdioxide, lactose, mannitol and other sugars, talc, milk protein,gelatin, starch, vitamins, cellulose and its derivatives, animal andvegetable oils, polyethylene glycols and solvents, such as sterilewater, alcohols, glycerol, and polyhydric alcohols. Intravenous vehiclesinclude fluid and nutrient replenishers. Preservatives includeantimicrobial, anti-oxidants, chelating agents, and inert gases. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike, as described, for instance, in Remington's PharmaceuticalSciences, 15th ed., Easton: Mack Publishing Co., 1405-1412, 1461-1487(1975), and The National Formulary XIV., 14th ed., Washington: AmericanPharmaceutical Association (1975), the contents of which are herebyincorporated by reference. The pH and exact concentration of the variouscomponents of the pharmaceutical composition are adjusted according toroutine skills in the art. See Goodman and Gilman's, The PharmacologicalBasis for Therapeutics (7th ed.).

The pharmaceutical compositions according to the disclosure may beadministered locally or systemically. A “therapeutically effective dose”is the quantity of an agent according to the disclosure necessary toprevent, to cure, or at least partially arrest a symptoms associatedwith a disease or disorder or to promote cell growth, proliferation ordifferentiation. Amounts effective for this use will, of course, dependon the severity of the disease, disorder, or desired effect and willdepend on weight and general state of the subject. Typically, dosagesused in vitro may provide useful guidance in the amounts useful for insitu administration of the pharmaceutical composition, and animal modelsmay be used to determine effective dosages for treatment of infections.Various considerations are described, e.g., in Langer, Science, 249:1527, (1990); Gilman et al. (eds.) (1990), each of which is hereinincorporated by reference. Dosages of pharmaceutically active compoundscan be determined by methods known in the art, see, e.g., Remington'sPharmaceutical Sciences (Maack Publishing Co., Easton, Pa.); Remington,The Science & Practice of Pharmacy, (Lippincott Williams & Wilkins;Twenty first Edition). The therapeutically effective dosage of anyspecific compound will vary somewhat from compound to compound, andpatient to patient, and will depend upon the condition of the patientand the route of delivery. As a general proposition, a dosage from about0.1 to about 100 mg/kg will have therapeutic efficacy, with all weightsbeing calculated based upon the weight of the compound, including thecases where a salt is employed. Toxicity concerns at the higher levelcan restrict intravenous dosages to a lower level such as up to about 10to about 20 mg/kg, with all weights being calculated based upon theweight of the compound, including the cases where a salt is employed. Adosage from about 10 mg/kg to about 50 mg/kg can be employed for oraladministration. Typically, a dosage from about 0.5 mg/kg to 15 mg/kg canbe employed for intramuscular injection. Particular dosages are about 1μmol/kg to 50 μmol/kg, and more particularly to about 22 μmol/kg and to33 μmol/kg of the compound for intravenous or oral administration,respectively.

In particular embodiments of the disclosure, more than oneadministration (e.g., two, three, four, or more administrations) can beemployed over a variety of time intervals (e.g., hourly, daily, weekly,monthly, etc.) to achieve therapeutic effects.

The compositions and chimera of the disclosure find use in veterinaryand medical applications. Suitable subjects include both avians andmammals, with mammals being preferred. The term “avian” as used hereinincludes, but is not limited to, chickens, ducks, geese, quail, turkeys,and pheasants. The term “mammal” as used herein includes, but is notlimited to, humans, bovines, ovines, caprines, equines, felines,canines, lagomorphs, etc. Human subjects include neonates, infants,juveniles, and adults. In other embodiments, the subject is an animalmodel of bone disease.

As used herein, “administering a therapeutically effective amount” isintended to include methods of giving or applying a pharmaceuticalcomposition of the disclosure to a subject that allow the composition toperform its intended therapeutic function.

The pharmaceutical composition can be administered in a convenientmanner, such as by injection (subcutaneous, intravenous, etc.), oraladministration, inhalation, transdermal application, or rectaladministration. Depending on the route of administration, thepharmaceutical composition can be coated with a material to protect thepharmaceutical composition from the action of enzymes, acids, and othernatural conditions that may inactivate the pharmaceutical composition.The pharmaceutical composition can also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof, and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. In all cases, the composition should besterile and should be fluid to the extent that easy syringabilityexists. The carrier can be a solvent or dispersion medium containing,for example, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), suitablemixtures thereof, and vegetable oils. The proper fluidity can bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size, in the case ofdispersion, and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be typical to includeisotonic agents, for example, sugars, polyalcohols, such as mannitol,sorbitol, or sodium chloride in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in thecomposition an agent that delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating thepharmaceutical composition in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the pharmaceutical composition into a sterilevehicle that contains a basic dispersion medium and the required otheringredients from those enumerated above.

The pharmaceutical composition can be orally administered, for example,with an inert diluent or an assimilable edible carrier. Thepharmaceutical composition and other ingredients can also be enclosed ina hard or soft-shell gelatin capsule, compressed into tablets, orincorporated directly into the individual's diet. For oral therapeuticadministration, the pharmaceutical composition can be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 1% by weightof active compound. The percentage of the compositions and preparationscan, of course, be varied and can conveniently be between about 5% toabout 80% of the weight of the unit.

The tablets, troches, pills, capsules, and the like can also contain thefollowing: a binder, such as gum gragacanth, acacia, corn starch, orgelatin; excipients such as dicalcium phosphate; a disintegrating agent,such as corn starch, potato starch, alginic acid, and the like; alubricant, such as magnesium stearate; and a sweetening agent, such assucrose, lactose or saccharin, or a flavoring agent such as peppermint,oil of wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it can contain, in addition to materials of the above type, aliquid carrier. Various other materials can be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules can be coated with shellac, sugar, or both.A syrup or elixir can contain the agent, sucrose as a sweetening agent,methyl and propylparabens as preservatives, a dye, and flavoring, suchas cherry or orange flavor. Of course, any material used in preparingany dosage unit form should be pharmaceutically pure and substantiallynon-toxic/biocompatible in the amounts employed. In addition, thepharmaceutical composition can be incorporated into sustained-releasepreparations and formulations.

Thus, a “pharmaceutically acceptable carrier” is intended to includesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the pharmaceutical composition, use thereof in thetherapeutic compositions and methods of treatment is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

In certain embodiments, the therapeutic method of the disclosureincludes administering the composition topically, systemically, orlocally as an implant or device. When administered, the therapeuticcomposition described by the disclosure are generally in a pyrogen-free,physiologically acceptable form. Further, the composition may desirablybe encapsulated or injected in a viscous form for delivery to the siteof bone, cartilage or tissue damage. Topical administration may besuitable for wound healing and tissue repair. Therapeutically usefulagents other than the chimeras of the disclosure may also optionally beincluded in the composition as described above, may alternatively oradditionally, be administered simultaneously or sequentially with thechimeras in the methods of the described herein. For example, preferablyfor bone and/or cartilage formation, the composition would include amatrix capable of delivering BMP chimeras or other therapeutic compoundsto the site of bone and/or cartilage damage, providing a structure forthe developing bone and cartilage and optimally capable of beingresorbed into the body. For example, the matrix may provide slow releaseof the BMP chimeras. Such matrices may be formed of materials presentlyin use for other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the subjectcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined, such as sintered hydroxyapatite, bioglass,aluminates, or other ceramics. Matrices may be comprised of combinationsof any of the aforementioned types of material, such as polylactic acidand hydroxyapatite or collagen and tricalciumphosphate. The bioceramicsmay be altered in composition, such as in calcium-aluminate-phosphateand processing to alter pore size, particle size, particle shape, andbiodegradability.

Certain compositions disclosed herein may be administered topically,either to skin or to mucosal membranes. The topical formulations mayfurther include one or more of the wide variety of agents known to beeffective as skin or stratum corneum penetration enhancers. Examples ofthese are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide,dimethylformamide, propylene glycol, methyl or isopropyl alcohol,dimethyl sulfoxide, and azone. Additional agents may further be includedto make the formulation cosmetically acceptable. Examples of these arefats, waxes, oils, dyes, fragrances, preservatives, stabilizers, andsurface active agents. Keratolytic agents such as those known in the artmay also be included. Examples are salicylic acid and sulfur.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.“Dosage unit form” as used herein, refers to physically discrete unitssuited as unitary dosages for the individual to be treated; each unitcontaining a predetermined quantity of pharmaceutical composition iscalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier. The specification for the dosageunit forms of the disclosure are related to the characteristics of thepharmaceutical composition and the particular therapeutic effect to beachieve.

The principal pharmaceutical composition is compounded for convenientand effective administration in effective amounts with a suitablepharmaceutically acceptable carrier in an acceptable dosage unit. In thecase of compositions containing supplementary active ingredients, thedosages are determined by reference to the usual dose and manner ofadministration of the said ingredients.

One of the challenges to using chimeras as therapeutics is the abilityto deliver the proteins effectively. The chimeras of the disclosure canbe delivered by several different methods. In the blood stream, thehalf-life of most TGF-β ligands is on the order of minutes. Tocompensate for the ligands being degraded so quickly, current therapiesinvolving TGF-β ligands use very high doses of the proteins.Alternatively, several means to directly modify the ligands or deliverysystems are available to help improve the stability or sustained releaseproperties of the ligands.

(1) Direct modification of the protein includes PEGylation as one commonform of modification. In this method, polyethylene glycol (PEG) iscovalently attached to the protein in hopes of improving stability byincreasing solubility, resistance to proteolysis, and decreasedimmunogenicity.

(2) Rational modification of residues on the protein surface. Byimproving any electrostatic instability, without changing overallprotein function, the overall stability of molecule can be improved.Using continuum electrostatic models, residues contributing toinstability can be located and then analyzed to see if it can be mutatedto a more favorable residue.

(3) Fusing the ligand to another protein or portion of a protein isanother technique to increase protein stability and solubility. Theantibody constant fragment (Fc) is common fusion partner used to improvethe stability and solubility.

(4) The use of liposomes can be used as a protein delivery vehicle.Liposomes are composed of any number of different phospholipids, whichself assemble to form spheres. The protein of interest is encapsulatedinside the bilayer, protecting it from the outside environment. Thephospholipid composition influences the exact properties of the liposomeand can be tailored to release the protein under any number of desiredconditions. Polymer/liposome composite systems are also available to beused as delivery systems. Ideally, this type of system combines theadvantages of each system to improve protein delivery.

(5) Similar to liposomes, polymers can be used as protein drug deliverysystems. The polymers are used to make a matrix, commonly what is termeda hydrogel due to the high water content of the material. The advantageof using the gel is it allows for long term, sustained release as wellas protecting the protein from proteolysis. As with the liposomes, thepolymers used to make the gel influence its properties. There are twogeneral classifications for the materials used to make the hydrogels:natural and unnatural polymers. Common materials used to createhydrogels using natural polymers include collagen, gelatin, fibrin,Hyaluronic acid, alginate, chitosan, and dextran. Synthetic polymersused to make hydrogels include Poly(ethylene oxide), Poly(acrylic acid),Poly(N-isopropylacrylamide), Poly(vinyl alcohol), and Polyphosphazene.

(6) A different kind of hydrogel can be created without the use ofpolymers, either natural or unnatural. Considered to be a bioactiveglass, or Xerogel, this material is created from silica and calciumphosphate layer capable of absorbing the protein of interest. See, e.g.,FIG. 8. The Xerogel increases the sustained release time of the proteinup to weeks. FIG. 1 shows results from cell viability assay usingosteoblast cell line MC3T3 by MTT assay, which shows that the xerogelmaterial is nontoxic up to the highest concentration of 30 mg/ml in theculture media we tested.

Chimera of the disclosure alone or in combination with apharmaceutically acceptable carrier can be used to treat any number ofdisease and disorder or modulate cellular or tissue activity.

The chimeric polypeptides of the disclosure can be used to treat anynumber of disease or disorders where modulating of TGF-beta activityprovides a therapeutic benefit. For example, the chimera of thedisclosure can be used in subjects suffering from osteoporosis,cartilage disease or periodontal diseases. The chimera can be used topromote bone and/or cartilage formation, inhibiting bone loss/density ordemineralization, promoting bone deposition and the like. Alternatively,the chimera can be used to inhibit excessive bone density and growth. Inother embodiment, the chimera can be used in the treatment of endocrinediseases and disorders, hyperparathyroidism, Cushing's disease,malabsorption, renal tubular acidosis, or thyrotoxicosis.

The chimera of the disclosure can also be used in the treatment ormodulating of sexual development, pituitary hormone production, andcreation of bone and cartilage. The chimera can also be used for thetreatment of cell proliferative diseases and disorders, cell growth anddifferentiation associated with inflammation, allergy, autoimmunediseases, infectious diseases, and tumors.

In a further aspect, the chimera of the disclosure can be used in thetreatment of neuromuscular disorders, such as muscular dystrophy andmuscle atrophy, congestive obstructive pulmonary disease, muscle wastingsyndrome, obesity or other metabolic diseases including, for example,type 2 diabetes.

The chimera of the disclosure can be used in degenerative musclediseases characterized by abnormal amount, development or metabolicactivity of muscle tissue, including gradual weakening and deteriorationof skeletal muscles. Examples of muscle disease and disorders include,but are not limited to, a muscle wasting disorder, cachexia, anorexia,AIDS wasting syndrome, muscular dystrophies, Duchenne Muscular Dystrophy(DMD), Becker Muscular Dystrophy (BMD), Myotonic Dystrophy (MMD) (alsoknown as Steinert's Disease), Oculopharyngeal Muscular Dystrophy (OPMD),Emery-Dreifuss Muscular Dystrophy (EDMD), Limb-Girdle Muscular Dystrophy(LGMD), Facioscapulohumeral Muscular Dystrophy (FSH or FSHD) (also knownas Landouzy-Dejerine), Congenital Muscular Dystrophy (CMD), and DistalMuscular Dystrophy (DD).

The chimera of the disclosure can be used in methods and compositions toprevent, treat, or alleviate symptoms of a neurodegenerative disease ordisorder including, but not limited to, Alzheimer's Disease (AD),Parkinson's Disease (PD), Amyotrophic Lateral Sclerosis (ALS), andHuntington's disease (HD), and other neuromuscular diseases, motorneuron diseases, diseases of the neuromuscular junction, and/orinflammatory myopathies.

A subject may have a disorder associated with abnormal cell growth anddifferentiation which may cause inflammation, allergy, autoimmunediseases, infectious diseases, and/or tumors. A subject may have a heartdisorder, such as a disorder associated with excessive cardiomyocyteproliferation or growth, or a disorder in which it would be desirable tostimulate cardiomyocyte growth or proliferation. Subject chimericTGF-beta superfamily proteins may be designed for the treatment ofessentially any disorder that is amenable to treatment by agonists orantagonists of a member of the TGF-beta superfamily.

The following examples are meant to further explain, but not limited theforegoing disclosure or the appended claims.

EXAMPLES Example 1

Generation of TGF-β Chimeras. To generate these novel TGF-β ligands, amodified directed evolution approach was utilized. Typically, thistechnique involves making a large number of random protein sequences,greater than 10³, either by mixing the sequences of homologous genes orinserting random mutations and then screening for the desired ligandproperties. In one set of experiments, sequences that were known torefold efficiently, termed the backbone ligand, were combined with asecond ligand sequence containing signaling properties desire to mimicthe target ligand. Using a structure guided approach, several TGF-βligand crystal structures were analyzed and divided into 6 distinctsections. These sections roughly encompass the following regions of theligand: section 1, N-terminus and beta strand 1; section 2, beta strand2; section 3, pre-helix loop; section 4, alpha helix; section 5, betastrand 3; and section 6, beta stand 4 and C-terminus. Using thisprotocol, 64 different ligand combinations are possible for each set ofTGF-β ligands chosen to be recombined. When two or more parental chainsare from different subfamilies (e.g. BMP/GDF v.s. TGFbeta), thedifference between their signaling mechanisms may not be captured ifsections 3 and 4 are separated. To be broadly applicable as the designprinciple, it is also part of the design to keep two structuralsegments, sections 3 and 4, can be treated as one section of either ofthe parental gene (referred to as section 3*4).

The strategy was implemented by making activin/BMP-2 chimeras usingactivin-βA as a target ligand and BMP-2 as the backbone ligand.Activin-βA was picked as the target ligand as it is biologically veryinteresting. BMP-2 was chosen as the backbone ligand because it has beenshown to refold with excellent efficiency, >10% dimer yield fromstarting denatured inclusion bodies, and these dimers have been shown tobe active in both in vitro and in vivo experiments. To design thevarious sections, a sequence alignment of BMP-2 and activin-βA wasperformed to locate regions of sequence identity between the ligands(FIG. 7). These regions were used as the boundaries for the differentsections. By using these parts of the sequence as the overlap regionsfor the oligonucleotides during PCR changes will not be introduced intoeither the BMP-2 or activin-βA sequences. The sequence alignment wasthen used in conjunction with data from previously solved BMP-2 andactivin-βA structures to ultimately determine the 6 sections (FIG. 7a-c). Due to limitations with regions of identity between the sequences,the sections had to be shifted slightly from ideal. Particularly, thepre-helix loop and the majority of the α-helix were combined into onesection, while the remainder of the α-helix to the beginning of betastrand 3 was placed into a different section (FIGS. 7 b and c).Additionally, 3-point mutations were inserted to allow for the cloningstrategy to be successful. At the end of section 3, the BMP-2 sequenceis TLVN, while the activin-βA sequence is TVIN (FIG. 7 a). Since theseresidue differences are conservative, the leucine and valine from theBMP-2 were introduced into the corresponding activin-βA sequence. Thethird mutation is found at the end of section 5. Here, the BMP-2sequence is LYLD, while the equivalent activin-βA sequence is LYYD (FIG.7 a). Since this residue difference is less conserved than the previoustwo, the tyrosine from activin-βA was inserted into the correspondingBMP-2 sequence.

The N-terminus of activin-βA contains 2 additional cysteines (FIG. 7 a)which form a 4^(th) intra-disulfide bond. To eliminate the potential ofthis extra disulfide bond complicating the refolding process, thesection which contained these residues was eliminated from section 1 ofactivin-βA chimera design.

For the activin/BMP-2 chimeras, the mature domains of human BMP-2 andhuman activin-βA were initially divided into 6 sections each and primerswere designed for each section. For BMP-2, the primers coded for thefollowing protein sequences: Section 1, QAKHKQRKRLKSSCKRHPLYVDFSDVGWND;Section 2, WIVAPPGYHAFYCHGECP; Section 3, FPLADHLNSTNHAIVQTLVN; Section4, SVNSKIPKACCVP; Section 5, TELSAISMLYYD; Section 6,ENEKVVLKNYQDMVVEGCGCR. For activin-βA, the primers coded for thefollowing protein sequences: Section 1, RGLECDGKVNICCKKQFFVSFKDIGWNDW;Section 2, WIIAPSGYHANYCEGECP; Section 3, SHIAGTSGSSLSFHSTLVN; Section4, HYRMRGHSPFANLKSCCVP; Section 5, TKLRPMSMLYYD; Section 6,DGQNIIKKDIQNMIVEECGCS. An overlapping PCR strategy was used to mix thevarious sections together to generate full length chimeras. To generatethe 1b chimeras, two oligos were used to insert the BMP-2 sequenceQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDII into the target gene. Outer primers forall constructs were constructed to incorporate a 5′ NdeI site and a 3′XhoI site for cloning into pET21a expression vector. The desired proteinsequences were confirmed by DNA sequencing.

The chimeras were labeled according to the sections they contained. Forexample, 1b2b3b4a5a6b, in which the b's represent that the section wastaken from BMP-2 and the a's represent that the section was derived fromactivin-βA. The chimeras were also given shorthand numeric designations,such as A/B2-020, so that any functional assays could be undertaken in ablind manner. Table 1 sets forth some of the various chimeras:

TABLE 1 BMP-2/activin Sample constructs Designation DNA Sequence ProteinSequence Exemplary Characteristics 1b2b3a4a5a6a AB2-001ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S S C K R HP L Y V D F S D Potential universalGTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N D W I V A P P G Y H A F YC H G E C P S H I antagonist because itAGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A G T S G S S L S F H S T L V N H YR M R G H S P F competes receptor bindingCCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC A N L K S C C V P T K L R P M S M LY Y D D G Q N I but not signaling. ActsCCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT I K K D I Q N M I V E E C G C S asneither BMP-2 nor CCTTCCACTCAACGTTGGTCAACCACTACCGCATGCG activin-βA.GGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGTGTCCCGACCAAGCTGAGACCCATGTCCATGTTGTACTATGATGATGGTCAAAACATCATCAAAAAGGACATTC AGAACATGATCGTGGAGGAGTGTGGGTGCTCA1b2b3a4a5b6a AB2-002 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q RK R L K S S C K R H P L Y V D F S D Activity in stem cellGTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N D W I V A P P G Y H A F YC H G E C P S H I differentiation assaysAGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A G T S G S S L S L H S T L V N H YR M R G H S P F unlike BMP-2. CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC A N LK S C C V P T E L S A I S M L Y Y D D G Q N I ICCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT K K D I Q N M I V E E C G C SCCTTACACTCAACGTTGGTCAACCACTACCGCATGCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGTGTCCCGACAGAGCTCAGTGCTATCTCGATGTTGTACTATGATGATGGTCAAAACATCATCAAAAAGGACATTC AGAACATGATCGTGGAGGAGTGTGGGTGCTCA1b2a3a4a5b6a AB2-003 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q RK R L K S S C K R H P L Y V D F S DGTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N D W I I A P S G Y H A N YC E G E C P S H I AGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A G T S G S S L SF H S T L V N H Y R M R G H S P F TCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC AN L K S C C V P T E L S A I S M L Y Y D D G Q N I ICCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT K K D I Q N M I V E E C G C SCCTTCCACTCAACGTTGGTCAACCACTACCGCATGCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGTGTCCCGACAGAACTCAGTGCTATCTCGATGTTGTACTATGATGATGGTCAAAACATCATCAAAAAGGACATTC AGAACATGATCGTGGAGGAGTGTGGGTGCTCA1b2a3b4b5a6a AB2-004 ATGCAAGCCAAACACAAACAGCGGAAGCGTCTTAAG M Q A K H K QR K R L K S S C K R H P L Y V D F S D ‘Super” BMP-2TCCAGCTGCAAAAGGCACCCTTTGTATGTGGACTTCA V G W N D W I I A P S G Y H A N YC D G E C P F P L activity, unable to beGTGATGTGGGGTGGAATGACTGGATCATTGCTCCCT A D H L N S T N H A I V Q T L V N SV N S K I P K A inhibited by Noggin CTGGCTATCATGCCAACTACTGCGACGGAGAATGCCC C V P T K L R P M S M L Y Y D D G Q N I I K K D ICTTTTCCTCTGGCTGATCATCTGAACTCCACTAATCA Q N M I V E E C G C STGCCATTGTTCAGACGTTGGTCAACTCTGTTAACTCTAAGATTCCTAAGGCATGCTGTGTCCCGACCAAGCTGAGACCCATGTCCATGTTGTACTATGATGATGGTCAAAACATCATCAAAAAGGACATTCAGAACATGATCGTG GAGGAGTGTGGGTGCTCA 1b2b3b4b5b6bAB2-005 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S SC K R H P L Y V D F S D AB2-005 (BMP-2_(mq)) (BMP-2_(mq))GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N D W I V A P P G Y H A F YC H G E C P F P L contains one amino acidAGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A D H L N S T N H A I V Q T L V N SV N S K I P K A (Met) added at the N-CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC C C V P T E L S A I S M L Y L D E NE K V V L K N Y terminus of mature BMP-2CCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC Q D M V V E G C G C R in nature.Met originates ATGCCATTGTTCAGACGTTGGTCAACTCTGTTAACTC from thetranslation TAAGATTCCTAAGGCATGCTGTGTCCCGACAGAACT initiation codon (ATG).CAGTGCTATCTCGATGCTGTACCTTGACGAGAATGA Unless it is truncatedAAAGGTTGTATTAAAGAACTATCAGGACATGGTTGT the N-terminus of AB2-005.GGAGGGTTGTGGGTGTCGC during the folding 1b2a3a4a5b6b AB2-006ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S S C K R HP L Y V D F S D Activity in stem cellGTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N D W I I A P S G Y H A N YC E G E C P S H I differentiation assaysAGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A G T S G S S L S F H S T L V N H YR M R G H S P F unlike BMP-2. TCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC A N LK S C C V P T E L S A I S M L Y L D E N E K VCCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT V L K N Y Q D M V V E G C G C RCCTTCCACTCAACGTTGGTCAACCACTACCGCATGCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGTGTCCCGACAGAACTCAGTGCTATCTCGATGTTGTACCTTGACGAGAATGAAAAGGTTGTATTAAAGAACTATC AGGACATGGTTGTGGAGGGTTGTGGGTGTCGC1b2a3a4a5a6b AB2-007 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q RK R L K S S C K R H P L Y V D F S D Activity in stem cellGTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N D W I I A P S G Y H A N YC E G E C P S H I differentiation assaysAGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A G T S G S S L S F H S T L V N H YR M R G H S P F unlike BMP-2. TCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC A N LK S C C V P T K L R P M S M L Y L D E N E K VCCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT V L K N Y Q D M V V E G C G C RCCTTCCACTCAACGTTGGTCAACCACTACCGCATGCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGTGTCCCGACCAAGCTGAGACCCATGTCCATGTTGTACCTTGACGAGAATGAAAAGGTTGTATTAAAGAACTAT CAGGACATGGTTGTGGAGGGTTGTGGGTGTCGC1b2a3a4a5a6a AB2-008 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q RK R L K S S C K R H P L Y V D F S D Functions like activin-GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N D W I I A P S G Y H A N YC E G E C P S H I βA in cell signaling andAGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A G T S G S S L S F H S T L V N H YR M R G H S P F in vivo experiments, ~4-TCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC A N L K S C C V P T K L R P M S M LY Y D D G Q N I fold lower potency;CCTTCCCATATAGCAGGCACGTCCGGGTCCTCACTGT I K K D I Q N M I V E E C G C Sreplaces TGF-beta 1 in CCTTCCATTCAACGTTGGTCAACCACTACCGCATGCGchemically-defined stem GGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGT cell mediacontaining GTCCCGACCAAGCTGAGACCCATGTCCATGTTGTACT FGF2.ATGATGATGGTCAAAACATCATCAAAAAGGACATTC AGAACATGATCGTGGAGGAGTGTGGGTGCTCA1b2a3a4a5a6a AB2-009 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q RK R L K S S C K R H P L Y V D F S D Functions like activin- L66V/V671GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N D W I I A P S G Y H A N YC E G E C P S H I βA in cell signalingAGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A G T S G S S L S F H S T V I N H YR M R G H S P F A and in vivo experiments,TCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC N L K S C C V P T K L R P M S M L YY D D G Q N I I ~10-fold lower potencyCCTTCCCATATAGCAGGCACGTCCGGGTCCTCACTGT K K D I Q N M I V E E C G C S inactivin-βA signaling CCTTCCATTCAACGGTGATCAACCACTACCGCATGCG activity.GGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGTGTCCCGACCAAGCTGAGACCCATGTCCATGTTGTACTATGATGATGGTCAAAACATCATCAAAAAGGACATTC AGAACATGATCGTGGAGGAGTGTGGGTGCTCA1b(1a_II)2a3a4a5a6a AB2-010 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A KH K Q R K R L K S S C K K Q F F V S F K D I Functions like activin-GTCCAGCTGTAAGAAACAGTTCTTTGTCAGTTTCAAG G W N D W I I A P S G Y H A N Y CE G E C P S H I A βA in cell signaling andGACATCGGGTGGAATGACTGGATCATTGCTCCCTCT G T S G S S L S F H S T L V N H Y RM R G H S P F A in vivo experiments, ~20-GGCTATCATGCCAACTACTGCGAGGGAGAATGCCCT N L K S C C V P T K L R P M S M L YY D D G Q N I I fold lower potency inTCCCATATAGCAGGCACGTCCGGGTCCTCACTGTCCT K K D I Q N M I V E E C G C Sactivin-βA signaling. TCCATTCAACGTTGGTCAACCACTACCGCATGCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGTGTCCCGACCAAGCTGAGACCCATGTCCATGTTGTACTATGATGATGGTCAAAACATCATCAAAAAGGACATTCAG AACATGATCGTGGAGGAGTGTGGGTGCTCA1b2b3b4b5a6a AB2-011 ATGCAAGCCAAACACCAACAGCGGAAACGCCTTAAG M Q A K H Q QR K R L K S S C K R H P L Y V D F S D ‘Super” BMP-2 activity,TCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTCA V G W N D W I V A P P G Y H A F YC H G E C P F P L unable to be inhibited byGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCCC A D H L N S T N H A I V Q T L V N SV N S K I P K A Noggin CGGGGTATCACGCCTTTTACTGCCACGGAGAATGCC C C V P T KL R P S M L Y Y D D G Q N I I K K D I QCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATCA N M I V E E C G C STGCCATTGTTCAGACGTTGGTCAACTCTGTTAACTCTAAGATTCCTAAGGCATGCTGTGTCCCGACCAAGCTGAGACCCTCCATGTTGTACTATGATGATGGTCAAAACATCATCAAAAAGGACATTCAGAACATGATCGTGGAG GAGTGTGGGTGCTCA 1b2b3b4b5b6aAB2-012 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S SC K R H P L Y V D F S D ‘Super” BMP-2 activity,GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N D W I V A P P G Y H A F YC H G E C P F P L partially inhibited byAGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A D H L N S T N H A I V Q T L V N SV N S K I P K A Noggin CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC C C V P T EL S A I S M L Y Y D D G R N I I K K D I QCCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC N M I V E E C G C SATGCCATTGTTCAGACGTTGGTCAACTCTGTTAACTCTAAGATTCCTAAGGCATGCTGTGTCCCGACAGAACTCAGTGCTATCTCGATGTTGTACTATGATGATGGTCGAAACATCATCAAAAAGGACATTCAGAACATGATCGTG GAGGAGTGTGGGTGCTCA 1b2b3b4b5a6bAB2-013 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S SC K R H P L Y V D F S D Activity comparable toGTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N D W I V A P P G Y H A F YC H G E C P E P L BMP-2, inhibited byAGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A D H L N S T N H A I V Q T L V N SV N S K I P K A Noggin CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC C C V P T KL R P M S M L Y Y D E N E K V V L K NCCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC Y Q D M V V E G C G C RATGCCATTGTTCAGACGTTGGTCAACTCTGTTAACTCTAAGATTCCTAAGGCATGCTGTGTCCCGACCAAGCTGAGACCCATGTCCATGTTGTACTATGATGAGAATGAAAAGGTTGTATTAAAGAACTATCAGGACATGGTTGT GGAGGGTTGTGGGTGTCGC 1b2a3b4b5a6bAB2-014 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S SC K R H P L Y V D F S D Activity comparable toGTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N D W I I A P S G Y H A N YC D G E C P F P L BMP-2, partially blockedAGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A D H L N S T N H A I V Q T L V N SV N S K I P K A by Noggin TCTGGCTATCATGCCAACTACTGCGACGGAGAATGC C C V P TK L R P M S M L Y L D E N E K V V L K N YCCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC Q D M V V E G C G C RATGCCATTGTTCAGACGTTGGTCAACTCTGTTAACTCTAAGATTCCTAAGGCATGCTGTGTCCCGACCAAGCTGAGACCCATGTCCATGTTGTACCTTGACGAGAATGAAAAGGTTGTATTAAAGAACTATCAGGACATGGTTGT GGAGGGTTGTGGGTGTCGC 1b2a3b4b5b6aAB2-015 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S SC K R H P L Y V D F S D ‘Super” BMP-2 activity,GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N D W I I A P S G Y H A N YC D G E C P F P L unable to be inhibited byAGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A D H L N S T N H A I V Q T L V N SV N S K I P K A Noggin TCTGGCTATCATGCCAACTACTGCGACGGAGAATGC C C V P T EL S A I S M L Y Y D D G Q N I I K K D I QCCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC N M I V E E C G C SATGCCATTGTTCAGACGTTGGTCAACTCTGTTAACTCTAAGATTCCTAAGGCATGCTGTGTCCCGACAGAACTCAGTGCTATCTCGATGTTGTACTATGATGATGGTCAAAACATCATCAAAAAGGACATTCAGAACATGATCGTG GAGGAGTGTGGGTGCTCA 1b,2a3b4b5b6bAB2-016 ATGCAAGCCAAACACAAACAGCGGAAGCGTCTTAAG M Q A K H K Q R K R L K S SC K R H P L Y V D F S D Activity comparable toTCCAGCTGCAAAAGGCACCCTTTGTATGTGGACTTCA V G W N D W I I A P S G Y H A N YC E G E C P F P L BMP-2, inhibited byGTGATGTGGGGTGGAATGACTGGATCATTGCTCCCT A D H L N S T N H A I V Q T L V N SV N S K I P K A Noggin CTGGCTATCATGCCAACTACTGCGAGGGAGAATGCC C C V P T EL S A I S M L Y L D E N E K V V L K N YCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATCA Q D M V V E G C G C RCGCCATTGTTCAGACGTTGGTCAACTCTGTTAACTCTAAGATTCCTAAGGCATGCTGTGTCCCGACAGAACTCAGTGCTATCTCGATGCTGTACCTTGACGAGAATGAAAAGGTTGTATTAAAGAACTATCAGGACATGGTTGTG GAGGGTTGCGGGTGTCGT 1b2b3b4a5a6aAB2-017 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S SC K R H P L Y V D F S D GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N DW I V A P P G Y H A F Y C H G E C P F P LAGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A D H L N S T N H A I V Q T L V N HY R M R G H S P CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC F A N L K S C C V PT K L R P M S M L Y Y D D G Q N CCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC II K K D I Q N M I V E E C G C S ATGCCATTGTTCAGACGTTGGTCAACCACTACCGCATGCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGTGTCCCGACCAAGCTGAGACCCATGTCCATGTTGTACTATGATGATGGTCAAAACATCATCAAAAAGGACATTCAGAACATGATCGTGGAGGAGTGTGGGTGCTCA 1b2b3b4a5b6b AB2-018ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S S C K R HP L Y V D F S D GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N D W I V AP P G Y H A F Y C H G E C P F P L AGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC AD H L N S T N H A I V Q T L V N H Y R M R G H S PCCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC F A N L K S C C V P T E L S A I S IL Y L D E N E K V CCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC V L K N Y Q D MV V E G C G C R ATGCCATTGTTCAGACGTTGGTCAACCACTACCGCATGCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGTGTCCCGACAGAACTCAGTGCTATCTCGATACTGTACCTTGACGAGAATGAAAAGGTTGTATTAAAGAACTATCAGGACATGGTTGTGGAGGGTTGTGGGTGTCGC 1b2b3b4a5a6b AB2-019ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S S C K R HP L Y V D F S D GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N D W I V AP P G Y H A F Y C H G E C P F P L AGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC AD H L N S T N H A I V Q T L V N H Y R M R G H S PCCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC F A N L K S C C V P T K L R P M S ML Y Y D E N E K CCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC V V L K N Y Q D MV V E G C G C R ATGCCATTGTTCAGACGTTGGTCAACCACTACCGCATGCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGTGTCCCGACCAAGCTGAGACCCATGTCCATGTTGTACTATGATGAGAATGAAAAGGTTGTATTAAAGAACTATCAGGACATGGTTGTGGAGGGTTGCGGGTGTCGT 1b2b3b4a5b6a AB2-020ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S S C K R HP L Y V D F S D GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N D W I V AP P G Y H A F Y C H G E C P F P L AGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC AD H L N S T N H A I V Q T L V N H Y R M R G H S PCCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC F A N L K S C C V P T E L S A I S ML Y Y D D G Q N I CCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC I K K D I Q N MI V E E C G C S ATGCCATTGTTCAGACGTTGGTCAACCACTACCGCATGCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGTGTCCCGACAGAACTCAGTGCTATCTCGATGTTGTACTATGATGATGGTCAAAACATCATCAAAAAGGACATTCAGAACATGATCGTGGAGGAGTGTGGGTGCTCA 1b2b3a4a5a6b AB2-021ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S S C K R HP L Y V D F S D GTCCAGCTGTAAGAGACACCCTTTGTATGTGGACTTC V G W N D W I V AP P G Y H A F Y C H G E C P S H I AGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC AG T S G S S L S F H S T L V N H Y R M R G H S P FCCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC A N L K S C C V P T K L R P M S M LY L D E N E K V CCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT V L K N Y Q D M VV E G C G C R CCTTCCACTCAACGTTGGTCAACCACTACCGCATGCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGTGTCCCGACCAAGCTGAGACCCATGTCCATGTTGTACCTTGACGAGAATGAAAAGGTTGTATTAAAGAACTAT CAGGACATGGTTGTGGAGGGTTGTGGGTGTCGC1b2b3a4a5b6b AB2-022 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q RK R L K S S C K R H P L Y V D F S DGTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N D W I V A P P G Y H A F YC H G E C P S H I AGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A G T S G S S L SF H S T L V N H Y R M R G H S P F CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC AN L K S C C V P T E L S A I S M L Y L D E N E K VCCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT V L K N Y Q D M V V E G C G C RCCTTCCACTCAACGTTGGTCAACCACTACCGCATGCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGTGTCCCGACAGAACTCAGTGCTATCTCGATGTTGTACTATGATGAGAATGAAAAGGTTGTATTAAAGAACTATC AGGACATGGTTGTGGAGGGTTGC1b2b3a4b5b6b AB2-023 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q RK R L K S S C K R H P L Y V D F S DGTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N D W I V A P P G Y H A F YC H G E C P S H I AGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A G T S G S S L SF H S T L V N S V N S K I P K A C C CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGCV P T E L S A I S M L Y L D E N E K V V L K N Y Q DCCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT M V V E G C G C RCCTTCCACTCAACGTTGGTCAACTCTGTTAACTCTAAGATTCCTAAGGCATGCTGTGTCCCGACAGAACTCAGTGCTATCTCGATGCTGTACCTTGACGAGAATGAAAAGGTTGTATTAAAGAACTATCAGGACATGGTTGTGGA GGGTTGCGGGTGTCGT 1b2b3a4b5b6aAB2-024 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S SC K R H P L Y V D F S D GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N DW I V A P P G Y H A F Y C H G E C P S H IAGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A G T S G S S L S F H S T L V N S VN S K I P K A C C CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC V P T E L S A I SM L Y Y D D G Q N I I K K D I Q N MCCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT I V E E C G C SCCTTCCACTCAACGTTGGTCAACTCTGTTAACTCTAAGATTCCTAAGGCATGCTGTGTCCCGACAGAACTCAGTGCTATCTCGATGTTGTACTATGATGATGGTCAAAACATCATCAAAAAGGACATTCAGAACATGATCGTGGAG GAGTGTGGGTGCTCA 1b2b3a4b5a6aAB2-025 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S SC K R H P L Y V D F S D GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N DW I V A P P G Y H A F Y C H G E C P S H IAGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A G T S G S S L S F H S T L V N S VN S K I P K A C C CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC V P T K L R P M SM L Y Y D D G Q N I I K K D I Q N CCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGTM I V E E C G C S CCTTCCACTCAACGTTGGTCAACTCTGTTAACTCTAAGATTCCTAAGGCATGCTGTGTCCCGACCAAGCTGAGACCCATGTCCATGTTGTACTATGATGATGGTCAAAACATCATCAAAAAGGACATTCAGAACATGATCGTGGAG GAGTGTGGGTGCTCA 1b2b3a4b5a6bAB2-026 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S SC K R H P L Y V D F S D GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N DW I V A P P G Y H A F Y C H G E C P S H IAGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A G T S G S S L S F H S T L V N S VN S K I P K A C C CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC V P T K L R P M SM L Y Y D E N E K V V L K N Y Q CCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT DM V V E G C G C R CCTTCCACTCAACGTTGGTCAACTCTGTTAACTCTAAGATTCCTAAGGCATGCTGTGTCCCGACCAAGCTGAGACCCATGTCCATGTTGTACTATGATGAGAATGAAAAGGTTGTATTAAAGAACTATCAGGACATGGTTGTGGA GGGTTGCGGGTGTCGT 1b2a3a4b5b6bAB2-027 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S SC K R H P L Y V D F S D GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N DW I I A P S G Y H A N Y C E G E C P S H IAGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A G T S G S S L S F H S T L V N S VN S K I P K A C C TCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC V P T E L S A I SM L Y L D E N E K V V L K N Y Q D CCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGTM V V E G C G C R CCTTCCACTCAACGTTGGTCAACTCTGTTAACTCTAAGATTCCTAAGGCATGCTGTGTCCCGACAGAACTCAGTGCTATCTCGATGTTGTACCTTGACGAGAATGAAAAGGTTGTATTAAAGAACTATCAGGACATGGTTGTGGA GGGTTGTGGGTGTCGC 1b2a3a4b5b6aAB2-028 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S SC K R H P L Y V D F S D GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N DW I I A P S G Y H A N Y C E G E C P S H IAGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A G T S G S S L S F H S T L V N S VN S K I P K A C C TCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC V P T E L N A I SM L Y Y D D G Q N I I K K D I Q N CCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGTM I V E E C G C S CCTTCCACTCAACGTTGGTCAACTCTGTTAACTCTAAGATTCCTAAGGCATGCTGTGTCCCGACAGAACTCAATGCTATCTCGATGTTGTACTATGATGATGGTCAAAACATCATTAAAAAGGACATTCAGAACATGATCGTGGAG GAGTGTGGGTGCTCA 1b2a3a4b5a6aAB2-030 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S SC K R H P L Y V D F S D GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N DW I I A P S G Y H A N Y C E G E C P S H IAGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A G T S G S S L S F H S T L V N S VN S K I P K A C C TCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC V P T K L R P M SM L Y Y D D G Q N I I K K D I Q N CCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGTM I V E E C G C S CCTTCCACTCAACGTTGGTCAACTCTGTTAACTCTAAGATTCCTAAGGCATGCTGTGTCCCGACCAAGCTGAGACCCATGTCCATGTTGTACTATGATGATGGTCAAAACATCATCAAAAAGGACATTCAGAACATGATCGTGGAG GAGTGTGGGTGCTCA 1b2a3b4a5a6aAB2-031 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S SC K R H P L Y V D F S D GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N DW I I A P S G Y H A N Y C E G E C P F P LAGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A D H L N S T N H A I V Q T L V N HY R M R G H S P TCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC F A N L K S C C V PT K L R P M S M L Y Y D D G Q N CCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC II K K D I Q N M I V E E C G C S ATGCCATTGTTCAGACGTTGGTCAACCACTACCGCATGCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGTGTCCCGACCAAGCTGAGACCCATGTCCATGTTGTACTATGATGATGGTCAAAACATCATCAAAAAGGACATTCAGAACATGATCGTGGAGGAGTGTGGGTGCTCA 1b2a3b4a5b6a AB2-032ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S S C K R HP L Y V D F S D GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N D W I I AP S G Y H A N Y C E G E C P F P L AGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC AD H L N S T N H A I V Q T L V N H Y R M R G H S PTCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC F A N L K S C C V P T E L S A I S ML Y Y D D G Q N I CCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC I K K D I Q N MI V E E C G C S ATGCCATTGTTCAGACGTTGGTCAACCACTACCGCATGCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGTGTCCCGACAGAACTCAGTGCTATCTCGATGCTGTACCTTGACGATGGTCAAAACATCATCAAAAAGGACATTCAGAACATGATCGTGGAGGAGTGTGGGTGCTCA 1b2a3b4a5b6b AB2-033ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S S C K R HP L Y V D F S D GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N D W I I AP S G Y H A N Y C E G E C P F P L AGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC AD H L N S T N H A I V Q T L V N H Y R M R G H S PTCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC F A N L K S C C V P T E L S A I S ML Y L D E N E K V CCTTTTCCTCTGGCTGATCATCTGAACTCTACTAATC V L K N Y Q D MV V E G C G C R ATGCCATTGTTCAGACGTTGGTCAACCACTACCGCATGCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGTGTCCCGACAGAACTCAGTGCTATCTCGATGCTGTACCTTGACGAGAATGAAAAGGTTGTATTAAAGAACTATCAGGACATGGTTGTGGAGGGTTGCGGGTGTCGT 1b2a3b4a5a6b AB2-034ATGCAAGCCAAACACAAACAGCGGAAGCGTCTTAAG M Q A K H K Q R K R L K S S C K R HP L Y V D F S D TCCAGCTGCAAAAGGCACCCTTTGTATGTGGACTTCA V G W N D W I I AP S G Y H A N Y C D G E C P F P L GTGATGTGGGGTGGAATGACTGGATCATTGCTCCCT AD H L N S T N H A I V Q T L V N H Y R M R G H S PCTGGCTATCATGCCAACTACTGCGACGGAGAATGCC F A N L K S C C V P T K L R P M S ML Y Y D E N E K CTTTTCCTCTGGCTGATCATCTGAACTCCACTAATCA V V L K N Y Q D MV V E G C G C R TGCCATTGTTCAGACGTTGGTCAACCACTACCGCATGCGGGGCCATAGCCCCTTTGCCAACCTCAAATCATGCTGTGTCCCGACCAAGCTGAGACCCATGTCCATGTTGTACTATGATGAGAATGAAAAGGTTGTATTAAAGAACT ATCAGGACATGGTTGTGGAGGGTTGCGGGTGTCGTBMP-2_(ma) BMP-2_(ma) ATGGCTCAAGCCAAACACAAACAGCGGAAACGCCTT M A Q A K H KQ R K R L K S S C K R H P L Y V D F S BMP-2_(ma) contains twoAAGTCCAGCTGTAAGAGACACCCTTTGTACGTGGAC D V G W N D W I V A P P G Y H A F YC H G E C P F P additional amino acidsTTCAGTGACGTGGGGTGGAATGACTGGATTGTGGCT L A D H L N S T N H A I V Q T L V NS V N S K I P K (Met-Ala) at the CCCCCGGGGTATCACGCCTTTTACTGCCACGGAGAA AC C V P T E L S A I S M L Y L D E N E K V V L K N N-terminal side ofTGCCCTTTTCCTCTGGCTGATCATCTGAACTCCACTA Y Q D M V V E G C G C R matureBMP-2 in nature ATCATGCCATTGTTCAGACGTTGGTCAACTCTGTTAA (QAKH . . . ). Metis CTCTAAGATTCCTAAGGCATGCTGTGTCCCGACAGA often truncated duringACTCAGTGCTATCTCGATGCTGTACCTTGACGAGAAT the folding process,GAAAAGGTTGTATTAAAGAACTATCAGGACATGGTT but Ala remainsGTGGAGGGTTGTGGGTGTCGC as the N-terminus of the final product. Eitherform is regarded as BMP-2_(ma). 1b_BMP7 NB2-BMP7ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S S C K R HP L Y V D F S D GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N D W I I AP E G Y A A Y Y C E G E C A F P L AGTGACGTGGGGTGGAATGACTGGATTATCGCGCCT NS Y M N A T N H A I V Q T L V H F I N P E T V P KGAAGGCTACGCCGCCTACTACTGTGAGGGGGAGTGT P C C A P T Q L N A I S V L Y F D DS S N V I L K K Y GCCTTCCCTCTGAACTCCTACATGAACGCCACCAACC R N M V V R A CG C H ACGCCATCGTGCAGACGCTGGTCCACTTCATCAACCCGGAAACGGTGCCCAAGCCCTGCTGTGCGCCCACGCAGCTCAATGCCATCTCCGTCCTCTACTTCGATGACAGCTCCAACGTCATCCTGAAGAAATACAGAAACATGGT GGTCCGGGCCTGTGGCTGCCAC 1b_BMP9NB2-BMP9 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S SC K R H P L Y V D F S D GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N DW I I A P K E Y E A Y E C K G G C F F P LAGTGACGTGGGGTGGAATGACTGGATTATTGCCCCA A D D V T P T K H A I V Q T L V H LK F P T K V G K AAAGAGTACGAGGCATACGAGTGTAAGGGCGGCTGT A C C V P T K L S PI S V L Y K D D M G V P T L K Y TTCTTTCCGCTGGCCGACGATGTCACCCCGACCAAGC HY E G M S V A E C G C R ACGCAATTGTCCAAACCTTAGTGCACCTGAAGTTCCCAACGAAAGTGGGTAAGGCATGTTGTGTGCCAACCAAGTTATCTCCAATTAGCGTGCTGTATAAGGATGATATGGGCGTGCCGACGTTAAAGTATCATTACGAGGGCATG AGCGTCGCAGAGTGTGGCTGCCGC 1b_GDF7NB2-GDF7 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S SC K R H P L Y V D F S D GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N DW I I A P L D Y E A Y H C E G L C D F P LAGTGACGTGGGGTGGAATGACTGGATTATCGCGCCG R S H L E P T N H A I I Q T L V N SM A P D A A P A S CTGGACTACGAGGCGTACCACTGCGAGGGCCTATGC C C V P A R L S PI S I L Y Y D A A N N V V Y K Q Y GATTTTCCTCTGCGTTCGCACCTCGAACCCACCAACCE D M V V E A C G C R ATGCCATCATTCAGACGTTGGTCAACTCCATGGCACCAGACGCGGCGCCGGCCTCCTGCTGTGTCCCGGCGCGCCTCAGCCCCATCAGCATCTTGTACTATGATGCCGCCAACAACGTTGTCTACAAGCAATACGAGGACATGGTG GTGGAGGCCTGTGGGTGTCGC 1b_GDF8NB2-GDF8 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S SC K R H P L Y V D F S D GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC V G W N DW I I A P K R Y K A N Y C S G E C E F V FAGTGACGTGGGGTGGAATGACTGGATTATTGCACCC L Q K Y P H T H L V H Q A N P R G SA G P C C T P T AAAAGATATAAGGCCAATTACTGCTCTGGAGAGTGT K M S P I N M L Y FN G K E Q I I Y G K I P A M V V GAATTTGTATTTTTACAAAAATACCCTCACACTCATC DR C G C S TTGTGCACCAAGCAAACCCCAGAGGTTCAGCAGGCCCCTGCTGTACTCCCACAAAGATGTCTCCAATCAATATGCTATATTTTAATGGCAAAGAACAAATAATATATGGGAAAATTCCAGCCATGGTAGTAGATCGCTGTGGGTG CTCA BMP2/BMP6 B2/B6 BMP-2_(wt) andBMP-6 are added BMP-2_(wt) sequence is reported above as AB2-005. BMP-6Has increased SMAD- together during the refolding to amino acidsequence. Two amino acids, MA, are mediated signaling generate theBMP2/BMP6 heterodimer. present in BMP-2_(wt) in contrast to matureactivity as compared to BMP-2_(wt) sequence herein is also form of BMP-2existent in nature. either BMP-2 or BMP-6. reported as AB2-005. BMP-6DNA M Q Q S R N R S T Q S Q D V A R V S S A S D Y N S S sequence is: E LK T A C R K H E L Y V S F Q D L G W Q D W I I AATGCAACAGAGTCGTAATCGCTCTACCCAGTCCCAG P K G Y A A N Y C D G E C S F P L NA H M N A T N GACGTGGCGCGGGTCTCCAGTGCTTCAGATTACAAC H A I V Q T L V H L MN P E Y V P K P C C A P T K L AGCAGTGAATTGAAAACAGCCTGCAGGAAGCATGA N A IS V L Y F D D N S N V I L K K Y R N M V V R AGCTGTATGTGAGTTTCCAAGACCTGGGATGGCAGGA C G C HCTGGATCATTGCACCCAAGGGCTATGCTGCCAATTACTGTGATGGAGAATGCTCCTTCCCACTCAACGCACACATGAATGCAACCAACCACGCGATTGTGCAGACCTTGGTTCACCTTATGAACCCCGAGTATGTCCCCAAACCGTGCTGTGCGCCAACTAAGCTAAATGCCATCTCGGTTCTTTACTTTGATGACAACTCCAATGTCATTCTGAAAAAATACAGGAATATGGTTGTAAGAGCTTGTGGATGCC AC

Protein Expression and Purification. The activin/BMP-2, 1b chimeras, andBMP-2_(ma) chimeras were expressed using a typical E. coli expressionsystem, and all 32 chimeras were found in the inclusion body fractions.The expressed inclusion bodies were isolated, purified, and refolded.The refolded ligands were purified using a Hi-trap heparin column (GEHealthcare) and reversed phase chromatography (GraceVydac). The ligandswere lyophilized and re-suspended in 4 mM HCl, pH 1 for use in all cellbased assays or 10 mM Na acetate, pH 4 for all biophysical assays.Activin-βA was expressed in a stably transfected CHO cell line andpurified using techniques known in the art. Noggin was expressed andpurified based on previously described protocols.

The activin/BMP-2 chimera inclusion bodies were seen as single bands ona reduced, SDS-PAGE gel and found at the expected size of ˜13 kDa (FIG.1 a). To standardize the refoldings, all activin/BMP-2 chimeras wererefolded in 100 mL volumes at a concentration of 50 mg/L. Theconcentration was chosen based on previously successful BMP-2, BMP-3,and GDF-5 refoldings. The volume was picked so that any dimer yield of2% or greater would generate enough protein for biophysical activityassays, yet small enough to be manageable with the large number ofsamples. Following refolding, the activin/BMP-2 samples were analyzedfor the formation of pure dimer, the desired product, after elution fromHeparin column (FIGS. 1 b and c). Surprisingly, all 32 activin/BMP-2samples showed the presence of some dimer and the chimeras were rankedbased on their refolding efficiency (dimer yield) and grouped into 4categories, from poor (<1%, −) to wild type (>10%, +++) (Table 2). To beclassified as a ‘successful’ chimera, the ligand needed to have arefolding efficiency equal to or greater than 5%. This efficiency wouldyield 2.5 mg/L of dimeric protein from a standard 1 L refolding at 50mg/L concentration, and would be considered suitable for experimentswhere large quantities are required, such as x-ray crystallography. Whenrefolding efficiency was calculated, 24 out 32 (75%) of theactivin/BMP-2 chimeras met this criteria (Table 2, supplemental, ++ or+++).

TABLE 2 Dimer Construct Name Yield Rating Rating System 1b2b3a4a5a6aAB2001 5% ++ +++ (wt) >10% 1b2b3a4a5b6a AB2002 7% ++ ++ 5-9%1b2a3a4a5b6a AB2003 1% − + 2-4% 1b2a3b4b5a6a AB2004 9% ++ −  <1%1b2b3b4b5b6b AB2005 >10% +++ 1b2a3a4a5b6b AB2006 9% ++ 1b2a3a4a5a6bAB2007 >10% +++ 1b2a3a4a5a6a AB2008 >10% +++ 1b2a3a4a5a6a AB2009 ~4% +L66V/V67I 1b(1a_II)2a3a4a5a6a AB2010 3% + 1b2b3b4b5a6a AB2011 >10% +++1b2b3b4b5b6a AB2012 >10% +++ 1b2b3b4b5a6b AB2013 >10% +++ 1b2a3b4b5a6bAB2014 >10% +++ 1b2a3b4b5b6a AB2015 >10% +++ 1b2a3b4b5b6b AB2016 >10%+++ 1b2b3b4a5a6a AB2017 5% ++ 1b2b3b4a5b6b AB2018 2% + 1b2b3b4a5a6bAB2019 3% + 1b2b3b4a5b6a AB2020 3% + 1b2b3a4a5a6b AB2021 6% ++1b2b3a4a5b6b AB2022 5% ++ 1b2b3a4b5b6b AB2023 3% + 1b2b3a4b5b6a AB20246% ++ 1b2b3a4b5a6a AB2025 4% + 1b2b3a4b5a6b AB2026 5% ++ 1b2a3a4b5b6bAB2027 >10% +++ 1b2a3a4b5b6a AB2028 4% + 1b2a3a4b5a6b AB2029 1% −1b2a3a4b5a6a AB2030 2% + 1b2a3b4a5a6a AB2031 4% + 1b2a3b4a5b6a AB20324% + 1b2a3b4a5b6b AB2033 4% + 1b2a3b4a5a6b AB2034 1% −

To be considered a successful ligand, the activin/BMP-2 chimeras notonly have to be refoldable but they also need to display signalingcharacteristics. To test for these properties, all activin/BMP-2chimeras, regardless of refolding efficiency, were initially subjectedto activin activity assays. Activin-like signaling characteristics weretested using a whole cell luciferase reporter assay sensitive toSmad-2/3 activation (as described below). Activin-βA is known to signalthrough and activate the Smad-2/3 pathway, so if any of theactivin/BMP-2 chimeras mimic activin-βA functionality, they shouldsignal in a similar manner. Out of all 32 chimeras, only 1, 1b2a3a4a5a6a(AB2-008), signaled in an activin-like manner. AB2-008 activates theluciferase reporter in a dose dependent manner similar to activin-βA.When the potency of the AB2-008 chimera was determined, the EC_(H) wascalculated to be 64.5 pM. This value is ˜2 fold lower than activin-βAwith an EC₅₀ of 28.8 pM. To confirm that the luciferase results were adirect response to Smad-2 activation, phospho-Smad-2 was tested in thepresence of AB2-008. Similar to activin-βA, the addition of AB2-008promotes an increase in phospho-Smad-2 levels. As expected, AB2-008,along with activin-βA, does not stimulate phospho-Smad-1 production,indicating only activation of a specific signaling pathway.Interestingly, AB2-008 exhibits BMP-2_(ma) refolding efficiencywith >10% dimer yield (Table 2).

To fully test if AB2-008 possesses complete activin-βA functionality,additional biophysical assays were performed. Cripto is a knownco-receptor for many of the TGF-β ligands and elicits a wide range ofresponses. For instance, the presence of Cripto is required for properNodal signaling, while it antagonizes TGF-β1 and activin signaling.Therefore, using the Smad-2/3 luciferase assay activin-βA and AB2-008signaling were monitored in the presence or absence of Cripto. In thepresence of Cripto, activin-βA signaling is decreased by ˜43% comparedactivin-βA alone. The AB2-008 chimera exhibits a similar decrease insignaling of ˜38% when Cripto is added to the assay. This resultconfirms that the AB2-008 chimera is a fully functioning activin mimicby being able to activate the activin signaling pathway as well havingthe ability to interact with other known activin binding partners.

Based on the results of AB2-008, 2 additional activin/BMP-2 chimeraswere generated to see if the potency of the chimeras could be increasedto wild type activin-βA levels. The first chimera, named 1b2a3a4a5a6aL66V/V67I (AB2-009), introduced a valine and iso-leucine into thechimera. These residues are found in the wild type sequence ofactivin-βA and were originally mutated to the corresponding BMP-2residues due to experimental design constraints (FIG. 7 a). The secondchimera, named 1b(1a_II)2a3a4a5a6a (AB2-010), replaces the second halfof the 1b section with the corresponding sequence from activin (FIG. 7a). This leaves only the 13 N-terminal residues preceding the firststructurally conserved cysteine, Leu-66, and Val-67 as components fromBMP-2 in this chimera construct. It is predicted that the introductionof additional activin residues into AB2-008 will improve its functionalcharacteristics (i.e. potency). AB2-009 and AB2-010 were expressed andrefolded as previously described for the other activin/BMP-2 chimeras.Unexpectedly, both of these new chimeras exhibited decreased refoldingefficiency compared to AB2-008. AB2-009 and AB2-010 saw a decreasefrom >10% dimer yield to ˜4% and ˜3% for AB2-009 and AB2-010,respectively (Table 2). While this result may not be surprising forAB2-010 since an 11 residue section was mutated, the drastic decreasefor AB2-009 was unexpected. Both the L66V and V67I mutations are veryconservative changes with only a 1 carbon difference between the sidechains of the mutated residues.

Following refolding, the new chimeras were subjected to the sameSmad-2/3 luciferase assay as AB2-008 previously. AB2-009 activated thereporter in a dose dependent manner and displayed activity comparable toAB2-008 with an EC₅₀ of 79.4 μM. However, while AB2-010 also activatedthe reporter, it showed a significant decrease in activity with anEC_(H) of 198.6 μM, or ˜3-fold weaker than AB2-008 and ˜7-fold weakerthan activin-βA. As with AB2-008, both AB2-009 and AB2-0010 showedSmad-2 phosphorylation. Since AB2-009 and AB2-010 did not show enhancedsignaling characteristics from AB2-008 in the luciferase assay, theywere not subjected to Cripto binding assay.

While the Smad-2/3 luciferase, Smad-2 phosphorylation, and Criptoreporter assays indicate that AB2-008, AB2-009, and AB2-010 signalthrough the activin pathway and function very similarly to activin-βA,these assays only show function in an in vitro setting. Therefore, morephysiologically relevant experiments are required to prove that theseactivin/BMP-2 chimeras will elicit a biological response similaractivin-βA. One classical method used to test for proper activinfunction is a follicle stimulating hormone (FSH) release assay. Ratanterior pituitary cells are known to release FSH in response to thepresence of activin in both in vivo and in vitro experiments. Therefore,rat anterior pituitary cells were exposed to increasing amounts ofactivin-βA or the activin/BMP-2 chimeras and FSH release was measured byradioimmunoassay. All three activin/BMP-2 chimeras showed a dosedependent increase in FSH release similar to activin-βA. The amount ofFSH release stimulated by the chimeras was decreased in the presence ofincreasing amounts of Inhibin. Combined with the in vitro assay results,the FSH release assay confirms that AB2-008, AB2-009, and AB2-010possess the complete activin-βA functional characteristics.

The chimeras were also tested to check for any additional signalingproperties. BMP-2 is already used as a therapeutic agent for certainbone treatments and having chimeras with altered BMP-2 function mayprove beneficial. To test if any of the activin/BMP-2 chimeras displayedunique signaling characteristics, a similar experiment to the activin-βAfunctional assay was performed. Here, a whole cell luciferase reporterassay sensitive to Smad-1/5 activation, the known BMP-2 signalingpathway, was used rather than a reporter sensitive to Smad-2/3activation. Monitoring the luciferase response in a dose dependantmanner, a number of activin/BMP-2 chimeras exhibit interesting traits.These activin/BMP-2 chimeras were identified and classified into 3groups: Those with upregulated or ‘super’ BMP-2 activity; those withinsensitivity to Noggin, a BMP-2 antagonist; or those with both ‘super’BMP-2 activity and insensitivity to Noggin. Activin/BMP-2 chimeras1b2a3b4b5a6a (AB2-004), 1b2b3b4b5a6a (AB2-011), 1b2b3b4b5b6a (AB2-012),and 1b2a3b4b5b6a (AB2-015) all fall into category of enhanced BMP-2activity with Noggin insensitivity. In the Smad-1 luciferase assay,these ligands activate the reporter at the same level as BMP-2_(wt)using 10× less protein (i.e., 10-fold higher activity). Grouped into thecategory of upregulated BMP-2 activity is 1b2b3b4b5a6b (AB2-013). Thischimera shows the same 10-fold increase in activity as AB2-004, -011,-012, -015, but the signal is decreased down to background levels uponthe addition of Noggin, similar to BMP-2_(wt). Chimera, 1b2a3b4b5a6b(AB2-014), fell into the final category of ligands with normal BMP-2signaling but with insensitivity. AB2-014 activates the luciferasereporter to the same level as BMP-2_(wt) but its signal cannot beblocked by the addition Noggin. AB2-008 was also tested to see if itactivated the Smad-1 pathway in addition to activating the Smad-2pathway. AB2-008 did not show any Smad-1 activation, even up to levelsof 1 μg/ml. This result confirms that AB2-008 is a specific activinmimic and does not exhibit non-specific signaling characteristics.

With the success of the AB2-008 chimera which refolds efficiently andpossesses activin-like signaling characteristics, the 1b section wasexamined as a general tool to improve the refolding of other currentlynon-refoldable TGF-β ligands. As mentioned before, the 1b section is 30a.a. long and comprises the N-terminus of BMP-2 as well as the residuesforming the first beta strand of finger 1 (FIG. 7 a). Based on analysisof the ternary structure of BMP-2/BMPRIa/ActRII, the majority of theresidues found in section 1b do not form any contacts with either theType I or Type II receptors. Indeed, of the few residues which dogenerate contacts with the Type I receptor, Val-26, Gly-27, and Trp-28,the tryptophan is invariant throughout the entire TGF-β superfamily,while the Gly-27 participates in a backbone interaction, and the valineis predominantly a non-polar amino acid at this position throughout theTGF-β superfamily. Based on this, it is possible that the 1b region,while not critical for contributing to ligand-receptor affinity andspecificity, is very helpful in proper disulfide bond formation duringthe chemical refolding process. Therefore, the 1b section was clonedinto the additional TGF-β ligands BMP-7, BMP-9, and GDF-8. As with theactivin/BMP-2 chimeras, the 1b chimeras were expressed in an E. coliexpression system and the inclusion bodies isolated to high purity.

Smad-1 Luciferase Assays in C2C12 Cells. Smad1-dependent luciferaseassays were performed using techniques known in the art. In brief, C2C12myoblast cells are cultured in Dulbecco's minimum essential medium(DMEM)+5% FBS supplemented with L-Glutamine and antibiotics. Forluciferase reporter assays, cells were trypsinized, washed twice withPBS and plated into 48-well plates with DMEM+0.1% FBS. Twenty four hourslater, cells were transfected with -1147Id1-luciferase constructcontaining the Smad binding sites (Id1-Luc), a Smad1 expressionconstruct, and a CAGGS-LacZ plasmid by using Fugene6 (Roche) accordingto the manufacturer's instruction and cells were stimulated withincreasing amounts of BMP-2_(ma) or the various activin/BMP-2 chimerasadded 24 hours post transfection. Luciferase activity was measured 24hours after stimulation with ligands and the values were normalized fortransfection efficiency by using beta-galactosidase activity. Theactivity of the luciferase reporter is expressed in fold-inductionrelative to control values that are obtained by using -927Id1-luciferasethat lacks Smad binding domains (Id1-Luc mut). To test for the abilityof Noggin to attenuate the Smad1 signaling of the ligands, theluciferase assays were repeated as described above, with a set dose ofNoggin included in the assay.

Smad-2 Luciferase Assays in HEK293 Cells. HEK293T cells were seeded into24-well plates coated with polylysine at a density of 150,000cells/well. After 24 h cells were transfected overnight with a mixtureof A3 Lux (25 ng) and β-galactosidase (25 ng) reporter plasmids, thetranscription factor FAST2 (50 ng), and empty pcDNA3 vector (400 ng)using Perfectin® transfection reagent (GenLantis) according to themanufacturer's recommendations. Then the cells were treated withincreasing doses of activin-βA or activin/BMP-2 chimeras for 16-24 h.The cells were harvested in ice-cold lysis buffer (1% Triton X-100 in 25mM glycylglycine, 4 nM EGTA, 15 mM MgSO₄ containing 1 mM dithiothreitol)and assayed for luciferase and β-galactosidase activities using standardmethods. To assess the ability of the activin/BMP-2 chimeras to bindknown TGF-β co-receptors, the HEK293T cells were treated with increasingdoses of activin-βA or activin/BMP-2 chimeras in the presence or absenceof transfected Cripto for 16-24 h (mouse Cripto construct was a generousgift from Malcolm Whitman (Department of Cell Biology, Harvard MedicalSchool, Boston, Mass.)). Activity was then measures as previouslydescribed.

Follicle Stimulating Hormone (FSH) Release from Rat Interior PituitaryCells. The assay was performed as previously described in the art.Briefly, freshly isolated cells from male Sprague-Dawley rat interiorpituitaries from several animals were combined and seeded into 96-wellplates at a density of 50,000 cells/well in βPJ medium supplemented with2% fetal bovine serum and appropriate growth factors. After 24 h cellswere treated with increasing doses of activin-βA or activin/BMP-2chimeras (0-40 nM). After 72 h, media were harvested and theconcentration of the secreted FSH was determined by radioimmunoassay.

Surface Plasmon Resonance (BIAcore) Affinity Studies. The affinity ofthe ligands to BMPRIa, ActRII, and ActRIIb was monitored by using aBiacore 3000 (GE Healthcare) and the data were analyzed by usingBIAevaluation software ver. 4.1 (GE Healthcare). Using primary aminecoupling, receptor ECDs were immobilized on a CM5 chip. The receptorswere immobilized independently on flow cells 2-4 for 10 minutes at aflow rate of 5 μL/min and a concentration of 20 μM in 10 mM Na acetate,pH 4.0. Flow cell 1 was left blank, no immobilized protein, as anegative control. The experiments were performed at a flow rate of 50μL/min in 20 mM Tris-HCl pH 7.9, 250 mM NaCl, 0.36%3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and0.005% Tween-20. A minimum of five concentrations, plus a zeroconcentration, were run per sample for kinetic analysis and the datawere fit by using a global 1:1 Langmuir binding with mass transfer.

Example 2

Synthesis of the BMP Heterodimer Ligand. The crystal structure ofBMP2-BMPRIa-ActRIIb has shown that each receptor molecule do notassociate extracellularly and have 4 distinct ligand-receptor interface.This suggested that a heterodimer would have 2 distinct type Iinterfaces and 2 distinct type II interfaces. To characterize functionaland other aspects of the BMP ligand recombinant heterodimers weresynthesized. The purity of the protein was verified by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). FIG. 1 aillustrates the migration of the BMP2/BMP6 heterodimer as a single bandunder non-reducing (lane 1) and as two distinct bands under reducingconditions (lane 3) on SDS-PAGE. The two distinct bands correlate to thetwo different monomer species (about 13 and 15 kDa respectively), of theBMP2/BMP6 heterodimer. This evidence is further supported withsurface-enhanced laser desorption/ionization time-of-flight massspectrometry (SELDI-TOF-MS) data. Three separate, purified samples ofthe BMP2 and BMP6 homodimers and the BMP2/BMP6 heterodimer were assayedon SELDI-TOF-MS. As FIG. 1 b demonstrates, the three samples eachcorrespond to their predicted mass with no other contaminating species.These assays indicate that a pure BMP2/BMP6 heterodimer was generated.

BMP Heterodimer Activity in vitro. To assay the interactions between theBMP2/BMP6 heterodimer and type I and type II TGF-beta receptor ECDs,surface plasmon resonance was utilized to measure the in vitro affinity.The TGF-beta receptors were immobilized to a chip and the TGF-betaligands were flowed over the surface while monitoring the interactions.Table 3 summarizes the ligands tested and the varying affinities for thetype I and type II receptor ECDs. In the case of the BMP2/BMP6heterodimer, it adopts the greater affinity from each of its BMP2 andBMP6 monomer subunits. As shown, the BMP2/BMP6 heterodimer has similaraffinity for the type I receptor as the BMP2 homodimer. However, insteadof adopting the type II receptor ECD affinity from the BMP2 subunit, theBMP2/BMP6 heterodimer has an affinity similar to the BMP6 homodimer forthe type II receptor. This indicates that the high affinity for the typeII receptor ECD is contributed by the BMP6 monomer subunit while thehigh affinity for the type I receptor ECD is contributed by the BMP2monomer subunit.

TABLE 3 Ligand Affinity Data from BIAcore Analysis Receptor BMPR-IaReceptor ActRIIb Ligand k_(off)[1/s]/k_(on)[1/M*s] K_(D) [nM] k_(off)[1/s]/k_(on)[1/M*s] K_(D) [nM] BMP2 1.11 × 10⁻³/8.52 × 10⁵ 1.31 2.57 ×10⁻²/6.68 × 10⁵ 38.5 BMP6 9.37 × 10⁻³/1.50 × 10⁵ 62.8 1.82 × 10⁻³/2.73 ×10⁵ 6.68 BMP2/BMP6 1.05 × 10⁻³/1.03 × 10⁶ 1.02     8.58/1.32 × 10⁹ 6.52Ligand affinity data from BIAcore experiments. The BIAcore data is shownas the dissociation rate, k_(off), and the association rate, k_(on),based on a global fit using the kinetic model 1:1 Langmuir binding withmass transfer. The binding constant K_(D) is calculated ask_(off)/k_(on). The receptors were immobilized to the chip surface, withthe ligands flowed over the surface.

To examine if receptor ECD affinity of the BMP2/BMP6 heterodimercorrelates with signaling activity, a luciferase reporter assay wasused. Using the C2C12 mouse myoblast cell line, the BMP ligands werequantitatively tested for the ability to activate a Smad1 dependentreporter gene. BMP2, BMP6 and BMP2/BMP6, all showed dose-dependentreporter activation. The BMP2/BMP6 heterodimer ligand showed furthergreater activation of the reporter gene than the BMP2 or BMP6 homodimercounterparts (FIG. 2). Up to 22-fold and 400-fold less BMP2/BMP6 wasrequired to activate the reporter gene to an equivalent level ascompared to the BMP2 and BMP6 homodimers respectively. Given thatBMP2/BMP6 heterodimer has a high affinity to both type I and type IIreceptor ECDs, the results suggest that increased affinity to receptorcorrelates with level of intracellular signaling activity.

To further characterize activities of BMP2/BMP6 heterodimer, an ex vivoassay using chick limb bud mesenchyme cells in micromass culture wasused. Primary cultured limb bud mesenchymal cells undergo chondrogenesisin a BMP-dependent manner, and this system allows the characterizationof homo and heterodimer in a biological process. FIG. 3 displaysmicrograph images of the staining of the chondrogenic nodules whoseformation is known to be stimulated by BMPs. The extent ofchondrogenesis is quantitated by measuring dye bound to the chondrogenicnodule. Similar to the reporter assay, a dose-dependent activation ofchondrogenesis of limb bud mesenchymal cells by different BMP ligands. Aunique aspect of this assay is that BMP6 has a slightly higher activitythan BMP2, while its activity in the Smad-1-dependent reporteractivation was significantly lower than that of BMP2. This likelyinvolves Type II receptor-initiated distinct signaling such as the p38pathway. Greater activity of BMP2/BMP6 heterodimer to activatechondrogenesis in this system was observed. BMP2/BMP6 activatedchondrogenesis to the similar level of BMP2 and BMP6 homodimer at10-fold concentration. The heterodimer ligand also induces a highermaximum response at the same concentration. This assay allows for thecorrelation of not only higher ligand-receptor affinity to highersignaling activity but extends this observation to an increase inbiological activity.

The data with ligand-receptor-ECD affinity, in vitro and ex vivo assayshave demonstrated that a functional asymmetric BMP2/BMP6 dimer wasgenerated. The asymmetric nature of the BMP2/BMP6 heterodimer allows forthe manipulation of specific TGF-beta receptor sites of the ligand. FIG.5 displays the specific mutagenesis of the BMP2/BMP6 heterodimer and thequantification of the ability of the different mutant variants toactivate a Smad1 dependent reporter gene using the same system asdescribed above (all factors are at 1 nM concentration). The quantifiedvalues are displayed as a percentage fold activation compared to theBMP2/BMP6 wild type heterodimer with no mutagenesis (normalized to 100%activation of the reporter gene). The BMP2/BMP6 heterodimer ligands withpoint mutations to only one of the two type I receptor interfaces withthe two type II receptor interfaces intact (FIG. 5, samples d and e),are able to activate the reporter gene, although between 20-80% comparedto the BMP2/BMP6 wild type heterodimer. In contrast, the BMP2/BMP6heterodimer with point mutations to only one of the two type II receptorinterfaces with the two type I interfaces intact (FIG. 5, sample f), cannot activate the reporter gene. The BMP2/BMP6 heterodimer ligands withpoint mutations disrupting one each of the two type I receptor and typeII receptor interfaces (FIG. 5, samples g and h), can not activate thereporter gene. These results demonstrate that 2 type II sites arerequired for signaling activity, while only 1 type I site was sufficientfor signaling. The difference of signaling activity between two ligandshaving 1 type I site mutation (d and e in FIG. 5) illustrates that Smad1activation directly correlates with affinity of a type I site and type Ireceptor (Table 3).

In further studies with the non-signaling BMP2/BMP6 heterodimer mutants,the ability of the ligands to bind receptor ECD was accessed. Despitethe inability of the ligand with only one active type II receptor siteto activate the reporter gene (FIG. 5, sample f), this ligand is stillable to bind type II receptor ECD under native-PAGE conditions. FIG. 6illustrates a type II receptor ECD saturation assay in which thereceptor ECD is run on the native-PAGE both in the absence and presenceof ligand. In comparing the lane with five micrograms of type IIreceptor ECD and no factor to the lane with ligand containing two activetype II receptor binding sites, the intensity of the band is diminishedby 9.0-fold, indicating that the receptor ECD has been incorporated in aligand-receptor complex. This represents about half of the receptor ECDincorporated into a ligand-receptor complex when comparing the intensityof three micrograms receptor ECD run alone. In the next lane, a ligandwith only one active type II receptor site shows an increase in the bandintensity by 3.3-fold compared to the ligand with two active type IIreceptor sites. And in the final lane, a ligand with no active type IIreceptor sites increases the band intensity by 1.8-fold compared to theligand with one active type II receptor site. As expected, the BMP2/BMP6ligand with one active type II receptor site falls in between the ligandwith two active type II receptor sites and that with no active type IIreceptor sites. The single intact type II receptor interface on themutated BMP2/BMP6 heterodimer is still able to bind one type II TGF-betareceptor ECD, indicating that the extracellular signaling complex canassemble. The inability of the mutated ligand to signal lies furtherdownstream from signaling complex assembly at the cell surface. Thisassay provides proof for an independent binding model of ligand-receptorcomplex formation where a single receptor ECD can bind to one of thefour receptor sites on the ligand regardless of the affinity orfunctionality of the other three receptor sites on the ligand.

The results from the analysis of the recombinant BMP heterodimers provethat a purified homogeneous heterodimer sample can be synthesized (FIG.1). The data further demonstrate that recombinant BMP heterodimers canbe expressed in E. coli as inclusion bodies, refolded, and purified at ascalable level.

The data obtained from the surface plasmon resonance affinity studies(Table 3), shows that a BMP heterodimer is more potent in vivo and invitro than the BMP homodimers. As compared to the homodimercounterparts, the BMP2/BMP6 heterodimer has the higher affinity receptorsites from each of its covalently linked monomer subunits. The BMP2/BMP6heterodimer has a high affinity type I receptor site comparable with theBMP2 homodimer and a high affinity type II receptor site comparable withthe BMP6 homodimer. Each of these homodimer ligands secondary receptorsites have lower affinity for the respective receptor compared to theirprimary receptor sites. This heterodimer ligand-receptor affinity data,for the first time, provides clear evidence for the mechanism of thehigh potency TGF-beta heterodimer ligands compared to their homodimercounterparts. With high affinity for both type I and type II receptorECDs, the TGF-beta signaling complex can more readily assemble andremain assembled as the cell surface. The augmented affinity of the BMPheterodimer correlates directly to increased signaling in the whole cellreporter assays (FIG. 2). Additionally, the ex vivo data from themesenchyme cell assays (FIGS. 3 and 4) demonstrates the ability of theBMP2/BMP6 heterodimer to be able to induce a response at a lowerconcentration and to a higher maximum than its homodimer counterparts.This data directly supports the correlation between increasedligand-receptor affinity, signaling activity, and biological activity.

While the high signaling activity of the BMP heterodimer was readilyachieved in the whole cell reporter system (FIG. 2), elucidating therequirements of the TGF-beta signaling complex and mechanism ofactivation proved much more difficult. The BMP heterodimer constructswith just one type I receptor site and two type II receptor sites, werestill able to activate the reporter gene, although to a lesser extentcompared to the fully functional BMP heterodimer (FIG. 5). However, theBMP heterodimer constructs with two type I receptor sites and only onetype II receptor site failed to activate the reporter gene (FIG. 5).This inconsistency between the number of required active type I and typeII receptor sites on the ligand can not be readily explained. The datademonstrating complex formation under native-PAGE conditions (FIG. 6),illustrates that the type II receptor ECD can bind to the mutatedheterodimer with only one active type II receptor interface. Thisindicates that the problem is not with signaling complex formationbetween ligand and receptor ECD at the cell surface. The complex withonly one type II receptor forms as readily as the complex with one typeI receptor, yet the complex with only one type II receptor does notinitiate downstream signaling.

The data suggest that a type II receptor kinase phosphorylates it'spartner type II receptor rather than itself, and such“cross-phosphorylation” is the molecular nature of theautophosphorylation. Alternatively, physical association ofintracellular kinase domain between two type II receptor is required for“auto phosphorylation”, although ECD does not associate one another.This initial step of the signaling cascade cannot occur in the absenceof one type II receptor, and thus, no signal is transduced. The abilityof some of the mutant BMP heterodimer ligands to form an activesignaling complex with only one type I receptor present (FIG. 5), occursbecause the two type II receptor kinases in the signaling complex areable to dimerize, autophosphorylate, and then transphosphorylate thesingle type I receptor kinase. No dimer of the type I receptor kinasesis required because the kinase domain is simply transphosphorylated bythe autophosphorylated dimer of type II receptor kinases.

The disclosure shows a truly independent ligand-receptor ECD bindingmodel for the signaling complex formation at the cell surface. As statedabove, mutated BMP2/BMP6 heterodimer ligand with only one active type IIreceptor site can still bind a single type II receptor ECD (FIG. 6).This evidence provides grounds for the assertion of the signalingcomplex's ability to form regardless of the affinity or functionality ofthe four individual receptor sites of the ligand. If each receptor siteon the ligand is able to bind in this independent fashion, another layerof complexity is added to the TGF-beta signaling complex formation. Withabout forty genes encoding for ligands and many functional ligandspossible because of heterodimers an intricate signaling mechanism mustexist. With the ability to only signal through twelve receptors, thisligand driven signaling mechanism must rely on the affinity of ligand'sindividual receptor sites to the different receptors. This is onlypossible if each of the four receptor sites on the ligand actindependently to recruit a receptor into the signaling complex. In orderto elicit different biological functions through the same twelvereceptors, the independent and individual affinities of each of the fourreceptor sites on the ligand is the key factor in fine tuning thebiological response. In this point of view, a role of specific ligand(homodimer or heterodimer) is to assemble distinct set of type I andtype II receptors with distinct affinity, which in turn, generatedifferent level of signaling and complexity of TGF-b signaling.

The scalable generation of a novel BMP2/BMP6 construct with highactivity in vitro and ex vivo has far reaching implications. Thismolecule served as the basis to determine the assembly of the TGF-betasuperfamily ligand-receptor signaling complex and to demonstrate thedirect correlation between ligand-receptor affinity, signaling activity,and biological activity. The differences in affinity between ligand andreceptor are crucial and the asymmetric heterodimer ligand signalingadds further complexity to the biological activity of the TGF-betamolecules. the study with the BMP heteromider illustrates how eachligand-receptor interaction contributes to the activity of the TGF-betasuperfamily.

Generation of heterodimer. The mature domains of human BMP2 (residues1-110) and human BMP6 (residues 1-132) were expressed in E. coli asinclusion bodies. Mutations to the wild type ligand sequences were basedon previously published findings which disrupt the ligand-receptorinterfaces (Keller et al., 2004; Kirsch et al., 2000). The expressedinclusion bodies were isolated, purified, and refolded. The refoldedBMP2 and BMP6 homodimers, and BMP2/BMP6 heterodimer were purified usinga HiTrap heparin column (GE Healthcare) and reversed phasechromatography (GraceVydac). The ligands were lyophilized andre-suspended in 10 mM sodium acetate pH 4.0. The ECDs of human BMPRIa(residues 1-129) and mouse ActRIIb (residues 1-98) were expressed in E.coli as thioredoxin fusion proteins. Mouse ActRII-ECD (residues 1-102)was expressed and purified from a P. pastoris expression system.

Surface plasmon resonance (BIAcore) affinity studies. The affinity ofthe ligands to BMPRIa, ActRII, and ACTRIIb was monitored by using aBiacore 3000 (GE Healthcare) and the data were analyzed by usingBIAevaluation software ver. 4.1 (GE Healthcare). Using primary aminecoupling, receptor ECDs were immobilized on a CM5 chip. The receptorswere immobilized independently on flow cells 2-4 for 10 minutes at aflow rate of 5 μL/min and a concentration of 20 μM in 10 mM sodiumacetate, pH 4.0. Flow cell 1 was left blank with no immobilized proteinas a negative control. The experiments were performed at a flow rate of50 μL/min in 20 mM Tris-HCl, pH 7.9, 250 mM NaCl, 0.36%3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), and0.005% Tween-20. At least five concentrations, plus a zeroconcentration, were run per sample for kinetic analysis and the datawere fit by using a global 1:1 Langmuir binding with mass transfer.

Luciferase reporter assays. Smad1-dependent luciferase assays wereperformed. In brief, C2C12 cells are cultured in Dulbecco's minimumessential medium (DMEM)+5% FBS supplemented with L-glutamine andantibiotics. For luciferase reporter assays, cells were trypsinized,washed twice with PBS, and plated into 48-well plates with DMEM+0.1%FBS. Twenty-four hours later, cells were transfected with-1147Id1-luciferase construct containing the Smad binding sites(Id1-Luc) (Nakashima et al., 2001), a Smad-1 expression construct, and aCAGGS-LacZ plasmid using Fugene6 (Roche) according to the manufacturer'sinstruction. Luciferase activity was measured 24 h after stimulationwith ligands, and the values were normalized for transfection efficiencyusing beta-galactosidase activity. The activity of the luciferasereporter is expressed in fold induction relative to control values thatare obtained using -927Id1-luciferase that lacks Smad1 binding domains(Id1-Luc mut).

Chick Limb Bud Micromass assays. Chick embryos at Hamburger-Hamiltonstage 23-24 (Hamburger and Hamilton, 1951) were collected in Hankssolution containing Ca2+ and Mg2+, and distal ⅓ part of limb buds weredissected out. Ectodermal sheets were removed by trypsinization (0.5% inHanks solution with Ca2+ and Mg2+) on ice for 30 min, and thenmesenchymal tissues were recovered and incubated in Ca²⁺ and Mg²⁺-freeHanks solution at 37° C. for 15 min. The mesenchyme cells weredissociated into single cells by pipetting in OptiMEM medium(Invitrogen) containing 1% FBS. Cultures were seeded into 96-well platesat 4×10⁵ cells/well. After 1 hour, media containing each ligand wasadded. Fresh media with ligands was changed daily, and cells wereanalyzed for chondrogenesis by Alcian blue staining to visualizecartilage nodule and quantification of chondrogenesis as described (Wadaet al., 2003).

Native-PAGE Ligand-Receptor ECD Complex Formation. Five micrograms ofpurified ActRII-ECD alone and with ten micrograms of BMP2/BMP6,BMP2/BMP6 with a single active type II receptor interface, or BMP2 withno active type II receptor interfaces was loaded onto a native-PAGE gelin 50 mM Tris-HCl, pH 7.9, 700 mM NaCl, and 1.8% CHAPS. The CoomassieBrilliant Blue (Bio-Rad) stained gel was analyzed using the “IntegratedDensity” function with NIH ImageJ software (Abramoff et al., 2004).

Example 3

(1) Development of Stem cell media (Valera et al., 2010) using AB2-008.Culturing human embryonic stem cells (hESC) in feeder free conditionsrequires the use of complex formulation media to maintain pluripotency.Unfortunately, commercial media are very expensive since the growthfactors required for the media are difficult to produce in mammaliancells. We have used mTeSR1 formulation to derive a new medium (CIVAmedium or mCIVA) for culturing human embryonic stem (hES) cells, andderiving and culturing induced pluripotent stem (iPS) cells (see FIG.2). CIVA medium substitutes TGFβ1 in mTeSR1 for AB2-008, a new chimericprotein with similar activity to Activin-A. hES cells cultured in thismedium on matrigel coating maintain pluripotent morphology for more than20 passages without karyotypic abnormalities. These cells are alsopositive for the pluripotency markers TRA-1-60 and SSEA-4, anddifferentiate in response to BMP-2 treatment. iPS cells cultured in thismedium also retain morphological characteristics of pluripotency andexpression of pluripotency markers. This new CIVA medium is alsosuitable to derivate iPS cells from human foreskin fibroblasts. CIVAmedium has all the properties desired of other commercial media for hEScells but can be formulated for considerably less cost than currentlyavailable media. FIG. 2. Development of mCIVA formulation using H9 hEScell line.

FIG. 9 shows H9 hES cells cultured in mCIVA using differentconcentrations of AB2-008 in the absence or presence of human FGF2. A.Differentiated H9 cells after 3 passages (1 ng/mL, AB2-008; no FGF2). B.Differentiated H9 cells after 3 passages (10 ng/mL, AB2-008; no FGF2).C. Differentiated H9 cells after 11 passages (100 ng/mL, AB2-008; noFGF2). D. Differentiated H9 cells after 12 passages (1 ng/mL, AB2-008;100 ng/mL, FGF2). E. H9 cells after 13 passages (10 ng/mL, AB2-008; 100ng/mL, FGF2). F. Differentiated H9 cells after 9 passages (100 ng/mL,AB2-008; 100 ng/mL, FGF2). Differentiated cells are denoted by arrows.

(2) Mineralization Data of AB2-004, AB2-011, AB2-015 (Yoon et al.,2010). Von Kossa staining was used to monitor the development ofpre-osteoblast cell line (MC3T3-E1) for extracellular mineralization byCa deposition. AB2-004 and AB2-011 show dramatic increase of the stainof nearly 10 times or more intensity. AB2-015 shows the most increasebut past 7 days period (not shown here). Control group (A) shows noappreciable mineralization, whereas the control ligand (BMP2) showsmodest increase of Ca flux and deposition as shown in Row B. of FIG. 10.

(3) Adult rat regeneration by AB2-004 (in collaboration) P3 digit ofAdult rat was severed. Either BMP2 or AB2-004 soaked in an agarose gelbead were added at the sight of surgery. Bone regrowth was monitored.BMP2-treated tissue shows no bone recovery. AB2-004-treated digit showsfull recovery.

(4) Smad2-based signaling (luciferase) assay by AB2-008, AB2-009,AB2-010 (Allendorph et al., 2010). AB2-008, AB2-009, and AB2-010activate the Smad2 pathway in a manner nearly undistinguishable fromactivin-βA. Potency for the chimeras is slightly reduced compared toactivin-βA, from 5 to 20-fold (see FIG. 11).

(5) Phospho-Smad-2 Assays by AB2-008, AB2-009, and AB2-010. Activin-βAspecifically phosphorylates Smad-2 and not Smad-1. This is in contrastto BMP-2 where we see specific phosphorylation of Smad-1 and not Smad-2.AB2-008, AB2-009, and AB2-010 display the same Smad-2 phosphorylationpattern as activin-βA. This confirms all three ligands stimulate theactivin-βA signaling pathway in a manner similar to activin-βA (see FIG.11).

(6) Follicle stimulating hormone release by AB2-008, AB2-009, andAB2-010. When activin-βA is added to rat interior pituitary cells, itcauses a dose dependent release of Follicle stimulating hormone (FSH).The activin-βA induced release of FSH can be blocked by the addition ofthe antagonist Inhibin. Similar to activin-βA, AB2-008, AB2-009, andAB2-010 cause the release of FSH and this stimulation is decreased inthe presence of Inhibin.

(7) Co-receptor binding with Cripto by AB2-008. Addition of Criptoreduces both activin-βA and AB2-008 signaling by comparable levels.These data indicate that AB2-008 possesses ability to bind Cripto,activin-βAco-receptor, confirming the functional similarity betweenAB2-008 and activin-βA.

(8) AB2-008 and Activin-βA Receptor Affinity

AB2-008 shows the receptor binding profile similar to that ofactivin-βA, which has high affinity to ActRII and none for BMPRIa.Further, the two ligands have nearly the same binding affinity. AB2-008is ˜1.7 weaker than activin-βA.

TABLE 4 Receptor affinity of Activin-βA versus AB2-008 Receptor AffinityData from BIAcore Experiments BMPRIa-ECD ActRII-ECD Ligandk_(off)[1/s]/k_(on)[1/M*s] K_(D) [nM] k_(off)[1/s]/k_(on)[1/M*s] K_(D)[nM] Activin-βA No Binding N.A. 7.16 × 10⁻⁴/3.52 × 10⁶ 0.203 AB2-008 NoBinding N.A. 8.24 × 10⁻⁴/2.39 × 10⁶ 0.344 The receptors were immobilizedto the chip surface with the ligands flowed over the surface. The datawere fit to a kinetic model (1:1 Langmuir binding with mass transfer) inwhich K_(D) is calculated as k_(off)/k_(on). The table reports data froma single trial. No Binding indicates that the interaction was notdetectable.

(9) Signaling activity of AB2-011, AB2-012, and AB2-015 by Smad-1pathway. AB2-004, AB2-011, AB2-012, and AB2-015 activate the Smad-1pathway more potently than BMP-2. This activation is 3 to 8-fold higherthan BMP-2 (FIG. 14).

(10) Receptor binding affinity of AB2-004, AB2-011, AB2-012, andAB2-015. AB2-004, AB2-011, AB2-012, and AB2-015 show similar bindingaffinity to ActRII as activin-βA. This is ˜100 fold higher than thebinding BMP-2 has for the same receptor. The type I receptor binding forthe AB chimeras ranges from near BMP-2 levels (AB2-004) to activin-βAlevels (no binding to BMPRIa for AB2-015).

TABLE 5 Receptor binding affinity of AB2-004, AB2-011, AB2-012, andAB2-015 Receptor Affinity Data from BIAcore Experiments BMPRIa-ECDActRII-ECD Ligand k_(off)[1/s]/k_(on)[1/M*s] K_(D) [nM]k_(off)[1/s]/k_(on)[1/M*s] K_(D) [nM] BMP-2 7.54 × 10⁻⁴/3.60 × 10⁵  2.094.05 × 10⁻²/1.10 × 10⁶ 36.8 Activin-βA No Binding N.A. 7.16 × 10⁻⁴/3.52× 10⁶ 0.203 AB2-004 1.85 × 10⁻²/1.13 × 10⁶ 16.4 1.87 × 10⁻⁴/4.91 × 10⁵0.381 AB2-011 1.93 × 10⁻²/4.07 × 10⁵ 47.4 3.39 × 10⁻⁴/6.46 × 10⁵ 0.525AB2-015 No Binding N.A. 3.71 × 10⁻⁴/1.61 × 10⁶ 0.230 AB2-012 8.48 ×10⁻²/1.61 × 10⁵ 526   1.60 × 10⁻³/3.39 × 10⁶ 0.472 The receptors wereimmobilized to the chip surface with the ligands flowed over thesurface. The data were fit to a kinetic model (1:1 Langmuir binding withmass transfer) in which K_(D) is calculated as k_(off)/k_(on). The tablereports data from a single trial. No Binding indicates that theinteraction was not detectable.

(11) Noggin insensitivity of AB2-004, AB2-011, AB2-012, and AB2-015.Noggin suppresses the signaling activity by directly complexing with theligand, and rendering it unable to bind its own receptors for signaling.In contrast to BMP-2 which is blocked to near background levels in thepresence of Noggin, the higher signaling of AB2-004, AB2-011, andAB2-015 are not inhibited by Noggin. AB2-012 is partially insensitive toNoggin Inhibition, with a ˜50% decrease in signaling with the additionof Noggin (FIG. 15). This property makes them particularly powerful intheir cellular signaling ability in vivo, including bone regeneration.

(12) Production Efficiency

TABLE 6 Production efficiency of refolding from E. coli inclusion body.Construct Dimer Yield Rating 1b2b3b4b5b6a >10%  +++ 1b2b3b4b5a6a >10% +++ 1b2b3b4b5a6b >10    +++ 1b2b3b4a5a6a 5% +++ 1b2b3b4a5b6b 2% +1b2b3b4a5a6b 3% + 1b2b3b4a5b6a 3% + 1b2b3a4a5a6a 5% ++ 1b2b3a4a5a6b 6%++ 1b2b3a4a5b6b 5% ++ 1b2b3a4a5b6a 7% ++ 1b2b3a4b5b6b 3% + 1b2b3a4b5b6a6% ++ 1b2b3a4b5a6a 4% + 1b2b3a4b5a6b 5% ++ 1b2a3a4a5a6a >10%  +++1b2a3a4a5a6b >10%  +++ 1b2a3a4a5b6b 9% ++ 1b2a3a4a5b6a >10%  +++1b2a3a4b5b6b >10%  +++ 1b2a3a4b5b6a 4% + 1b2a3a4b5a6b 1% − 1b2a3a4b5a6a2% + 1b2a3b4b5b6b >10%  +++ 1b2a3b4b5b6a >10%  +++ 1b2a3b4b5a6a 9% ++1b2a3b4b5a6b >10%  +++ 1b2a3b4a5a6a 4% + 1b2a3b4a5b6a 4% + 1b2a3b4a5b6b4% + 1b2a3b4a5a6b 1% − 1b2a3a4a5a6a L66V/V67I 4% + 1b(1a_II)2a3a4a5a6a3% +

(13) Receptor binding affinity of BMP2/BMP6 Heterodimer (Isaacs et al.,2010). BMP2/BMP6 heterodimer has the binding characteristics of BMP2 fortype I receptor BMPRIa and BMP6 for the type II receptor ActRIIb.Maintaining high affinity for each receptor type by the heterodimerligand makes BMP2/BMP6 heterodimer stronger in signaling activities thanits homodimeric counterparts, BMP2 and BMP6 homodimers (Table 7).

TABLE 7 Receptor binding affinity measured by Surface plasmon resonance.Receptor BMPRIa K_(D) Receptor ActRIIb K_(D) Ligandk_(off)[1/s]/k_(on)[1/M*s] [nM] k_(off)[1/s]/k_(on)[1/M*s] [nM] BMP21.11 × 10⁻³/8.52 × 10⁵ 1.31 2.57 × 10⁻²/6.68 × 10⁵ 38.5 BMP6 9.37 ×10⁻³/1.50 × 10⁵ 62.8 1.82 × 10⁻³/2.73 × 10⁵ 6.68 BMP2/ 1.05 × 10⁻³/1.03× 10⁶ 1.02 8.61 × 10⁻³/1.32 × 10⁶ 6.52 BMP6

(14) SMAD-1 Signaling activity of BMP2/BMP6 Heterodimer. The BMP2/BMP6heterodimer is much more active than either BMP2 or BMP6 alone.BMP2/BMP6 has an EC50 that is at least an order of magnitude higher thanBMP2 or BMP6 alone. Further, the maximal response reached by BMP2/BMP6is higher than the combination of maximum signal reached by BMP2 andBMP6 alone.

(15) Chick Limb Bud Micromass assays for BMP2/BMP6 heterodimer.BMP2/BMP6 induces chondrogenesis more potently than either BMP2 or BMP6homodimers. In chick limb bud mesenchyme cell micromass culturechrondogenesis assays, after three days we see that BMP2/BMP6 induceschondrogenesis at both lower concentrations and to a higher level thaneither BMP2 or BMP6.

Example 4

Description of subdomains (building blocks) for generating DesignerLigand. In order to create the chimeras, a first step was deciding whereto make the borders for each of the segments. The chimera library hasbeen constructed using activin-βA and BMP-2 as two sequence sources. Todesign the cut-off regions (Junction) for the sections to make theactivin/BMP-2 (AB) chimera, a structure-guided approach combined withprotein sequence alignment was used. Initially, the 3-dimensionalcrystal structures of activin-βA (Harrington et al., 2006) and BMP-2(Allendorph et al., 2006) were inspected structurally. From thisanalysis, the ligands were loosely divided into 6 distinct sections (seeFIG. 7 for segments 1 through 6). The exact segment junctions wereultimately determined following a protein sequence alignment of the twoligands to minimize any sequence changes of either protein sequence as aresult of joining the Junction. Further, the segmental boundaries werechosen to be located in structural regions away from receptor bindingsites.

Detailed descriptions of Junctions: Between segments 1 and 2 (Junction1): Focusing on the boundary of segment 1 and segment 2, we found a10-residue region that is highly conserved between BMP-2 and activin-βA.Indeed, 8 of the 10 residues are identical and the other two are veryconservative differences. This area is located in the tip region ofFinger 1 and depending of the ligand, makes or is predicted to makelimited contacts with either receptor type. Based on the ternary crystalstructure of BMP-2/BMPRIa/ActRII (Allendorph et al., 2006), only Val-26,Gly-27, and Trp-28 (BMP-2 numbering) generate contacts with the type Ireceptor. Of these three residues, only Val-26 is different between theligands, but it is a very conservative change since the correspondingresidue in activin-βA is Ile-23. Since the residues in this region arevery similar and not involved in receptor binding, it makes for a goodboundary point for segment 1 and 2.

Between segments 2 and 3 (Junction 2): Moving to the boundary regionbetween segments 2 and 3, another good area for our boundary cut-off canbe found. Here, a 4-residue sequence that is identical betweenactivin-βA and BMP-2 exists. When the ligands are properly folded, thisregion is located in the center of the dimer, with both cysteinesparticipating in the cystine knot. This is advantageous because theresidues here are buried from the surface of the ligands and do notparticipate in any ligand-receptor interactions.

Between segments 4 and 5 (Junction 4): Similar to the segment 2/3boundary, the segment 4/5 boundary is situated in an excellent locationfor the cut-off. Here, we find a 5-residue region of sequence identityand, as with the segment 2/3 boundary, this region is buried at thecenter of the ligand dimer. The 2 cysteine residues participate in boththe cystine knot as well as the inter-monomer disulfide bond. Again,this location prevents the residues in this region from participating inreceptor binding interactions.

Between segments 5 and 6 (Junction 5): To extend the design of BMP-2 andactivin-βA chimeras, other boundary regions have been chosen tofacilitate generating RASCH constructs using all members of the TGF-βsuperfamily. Along with sharing structural architecture, the TGF-βsuperfamily ligands seem to have certain regions in their proteinsequences that are highly conserved. Interestingly, these regionscoincide with the boundary regions chosen for making the BMP-2 andactivin-βA chimeras. For example, in the boundary region of 4 and 5,most ligands share 3 out of the 4 residues that define the boundarydomain. This high degree of similarity, coupled with these regions beingisolated from the receptor binding sites, indicates RASCH as theuniversal strategy to create a library of Designer Ligands with newfunctionalities.

Between segments 3 and 4 ((Junction 3): The boundary between segments 3and 4 is subject to structural variability between differentsubfamilies, in which ligand-receptor assembly mechanism can differsubstantially. In that regard, segments 3 and 4 can be treated as onesegmental piece such that two segments will be derived from the commonparental strand to preserve their structural integrity.

The structural similarity among all TGF-beta superfamily ligands formsthe rational basis for designing chimeric protein by exchanging(swapping) related segments of the sequences known to carry out certainfunctionality such as molecular recognition. Protein engineering ofAntibody chain, or more specifically of antibody fragment (Fab), will bea prime example where the basic structural scaffold is built on the Corearchitecture of the light- and heavy chain sequences, for which sixvariable loops, three from each of the two chains, are responsible forthe role of epitope-binding specificity. In the similar vein, theTGF-beta superfamily ligands share their structural framework as abutterfly-like architecture. A portion(s) of the sequence segmentsfunctionally equivalent to variable loop regions of Antibody can then be‘implanted’ to transfer recognition specificity from one ligand toanother. Our design principle distinguishes itself from theaforementioned ‘functional transfer by sequence implantation’. The newchimeric library is created on the basis of structural feasibility ofeach subdomain as defined by each Junction. The junctions between thevarious domains of the TGF-beta family members used to generate thechimeras of the disclosure provide useful building blocks of the chimeralibrary. By this reasoning, Junctions 1, 2, 4, and 5 are well defined tobe broadly applicable to all TGF-beta superfamily members, whereasJunction 3 is not broadly applicable. The application of Junction 3 inthe chimera design depends on the target sequences, in which casesubdomain segments 3 and 4 can be treated instead as one segment indesigning the chimera library. The approach maximizes the chance ofproducing such protein products that are foldable, for which functionalcharacterization will then follow.

Table of additional sequences: Protein TGF-β Ligand DNA SequenceSequence BMP-2 caagccaaacacaaacagcggaaacgccttaagtccagctgtaQAKHKQRKRLKSSCKRHPL agagacaccctttgtacgtggacttcagtgacgtggggtggaatYVDFSDVGWNDWIVAPPG gactggattgtggctcccccggggtatcacgccttttactgccacYHAFYCHGECPFPLADHLN ggagaatgcccttttcctctggctgatcatctgaactccactaatcSTNHAIVQTLVNSVNSKIPK atgccattgttcagacgttggtcaactctgttaactctaagattcctACCVPTELSAISMLYLDENE aaggcatgctgtgtcccgacagaactcagtgctatctcgatgctKVVLKNYQDMVVEGCGCR gtaccttgacgagaatgaaaaggttgtattaaagaactatcagga (SEQ IDNO: 2) catggttgtggagggttgtgggtgtcgc (SEQ ID NO: 1) BMP-3cagtggattgaacctcggaattgcgccaggagatacctcaaggt QWIEPRNCARRYLKVDFAD(osteogenin) agactttgcagatattggctggagtgaatggattatctcccccaaIGWSEWIISPKSFDAYYCSG gtcctttgatgcctattattgctctggagcatgccagttccccatgcACQFPMPKSLKPSNHATIQS caaagtctttgaagccatcaaatcatgctaccatccagagtatagIVRAVGVVPGIPEPCCVPEK tgagagctgtgggggtcgttcctgggattcctgagccttgctgtgMSSLSILFFDENKNVVLKV taccagaaaagatgtcctcactcagtattttattctttgatgaaaatYPNMTVESCACR(SEQ ID aagaatgtagtgcttaaagtataccctaacatgacagtagagtctt NO:43) gcgcttgcaga (SEQ ID NO: 42) BMP-4(BMP-agccctaagcatcactcacagcgggccaggaagaagaataag SPKHHSQRARKKNKNCRR 2b)aactgccggcgccactcgctctatgtggacttcagcgatgtggg HSLYVDFSDVGWNDWIVActggaatgactggattgtggccccaccaggctaccaggccttct PPGYQAFYCHGDCPFPLADactgccatggggactgcccctttccactggctgaccacctcaac HLNSTNHAIVQTLVNSVNStcaaccaaccatgccattgtgcagaccctggtcaattctgtcaatt SIPKACCVPTELSAISMLYLccagtatccccaaagcctgttgtgtgcccactgaactgagtgcc DEYDKVVLKNYQEMVVEGatctccatgctgtacctggatgagtagataaggtggtactgaaaa CGCR(SEQ ID NO: 45)attatcaggagatggtagtagagggatgtgggtgccgc (SEQ ID NO: 44) BMP-5gcagccaacaaacgaaaaaatcaaaaccgcaataaatccagct AANQNRNKSSSHQDSSRMSctcatcaggactcctccagaatgtccagtgttggagattataaca SVGDYNTSEQKQACKKHELcaagtgagcaaaaacaagcctgtaagaagcacgaactctatgt YVSFRDLGWQDWIIAPEGYgagcttccgggatctgggatggcaggactggattatagcacca AAFYCDGECSFPLNAHMNAgaaggatacgctgcattttattgtgatggagaatgttcttttccactt TNHAIVQTLVHLMFPDHVPaacgcccatatgaatgccaccaaccacgctatagttcagactct KPCCAPTKLNAISVLYFDDSggttcatctgatgtttcctgaccacgtaccaaagccttgttgtgctc SNVILKKYRNMVVRSCGCHcaaccaaattaaatgccatctctgttctgtactttgatgacagctcc (SEQ ID NO: 47)aatgtcattttgaaaaaatatagaaatatggtagtacgctcatgtg gctgccac(SEQ ID NO: 46)BMP-6(Vgr-1) caacagagtcgtaatcgctctacccagtcccaggacgtggcgcQQSRNRSTQSQDVARVSSA gggtctccagtgcttcagattacaacagcagtgaattgaaaacaSDYNSSELKTACRKHELYV gcctgcaggaagcatgagctgtatgtgagtttccaagacctgggSFQDLGWQDWIIAPKGYAA atggcaggactggatcattgcacccaagggctatgctgccaattNYCDGECSFPLNAHMNAT actgtgatggagaatgctccttcccactcaacgcacacatgaatNHAIVQTLVHLMNPEYVPK gcaaccaaccacgcgattgtgcagaccttggttcaccttatgaaPCCAPTKLNAISVLYFDDNS ccccgagtatgtccccaaaccgtgctgtgcgccaactaagctaaNVILKKYRNMVVRACGCH atgccatctcggttctttactttgatgacaactccaatgtcattctga (SEQID NO: 49) aaaaatacaggaatatggttgtaagagcttgtggatgccac (SEQ ID NO: 48)BMP-7(OP-1) tccacggggagcaaacagcgcagccagaaccgctccaagacSTGSKQRSQNRSKTPKNQE gcccaagaaccaggaagccctgcggatggccaacgtggcagALRMANVAENSSSDQRQA agaacagcagcagcgaccagaggcaggcctgtaagaagcacCKKHELYVSFRDLGWQDW gagctgtatgtcagcttccgagacctgggctggcaggactggatIIAPEGYAAYYCEGECAFPL catcgcgcctgaaggctacgccgcctactactgtgagggggagNSYMNATNHAIVQTLVHFI tgtgccttccctctgaactcctacatgaacgccaccaaccacgcNPETVPKPCCAPTQLNAISV catcgtgcagacgctggtccacttcatcaacccggaaacggtgLYFDDSSNVILKKYRNMVV cccaagccctgctgtgcgcccacgcagctcaatgccatctccgt RACGCH(SEQ ID NO: 51) cctctacttcgatgacagctccaacgtcatcctgaagaaatacagaaacatggtggtccgggcctgtggctgccac (SEQ ID NO: 50) BMP-8(OP-2)gcagtgaggccactgaggaggaggcagccgaagaaaagcaa AVRPLRRRQPKKSNELPQAcgagctgccgcaggccaaccgactcccagggatctttgatgac NRLPGIFDDVHGSHGRQVCgtccacggctcccacggccggcaggtctgccgtcggcacgag RRHELYVSFQDLGWLDWVIctctacgtcagcttccaggacctcggctggctggactgggtcat APQGYSAYYCEGECSFPLDcgctccccaaggctactcggcctattactgtgagggggagtgct SCMNATNHAILQSLVHLMKccttcccactggactcctgcatgaatgccaccaaccacgccatc PNAVPKACCAPTKLSATSVctgcagtccctggtgcacctgatgatgccagacgcagtcccca LYYDSSNNVILRKHRNMVVaggcgtgctgtgcacccaccaagctgagcgccacctctgtgct KACGCH (SEQ ID NO: 53)ctactatgacagcagcaacaatgtcatcctgcgcaagcaccgcaacatggtggtcaaggcctgcggctgccac (SEQ ID NO: 52) BMP-9(GDF-agcgccggggctggcagccactgtcaaaagacctccctgcgg SAGAGSHCQKTSLRVNFED 2)gtaaacttcgaggacatcggctgggacagctggatcattgcacc IGWDSWIIAPKEYEAYECKcaaggagtatgaagcctacgagtgtaagggcggctgcttcttcc GGCFFPLADDVTPTKHAIVccttggctgacgatgtgacgccgacgaaacacgctatcgtgca QTLVHLKFPTKVGKACCVPgaccctggtgcatctcaagttccccacaaaggtgggcaaggcc TKLSPISVLYKDDMGVPTLtgctgtgtgcccaccaaactgagccccatctccgtcctctacaag KYHYEGMSVAECGCR (SEQgatgacatgggggtgcccaccctcaagtaccattacgagggca ID NO: 55)tgagcgtggcagagtgtgggtgcaggtag (SEQ ID NO: 54) BMP-10aacgccaaaggaaactactgtaagaggaccccgctctacatcg NAKGNYCKRTPLYIDFKEIGacttcaaggagattgggtgggactcctggatcatcgctccgcct WDSWIIAPPGYEAYECRGVggatacgaagcctatgaatgccgtggtgtttgtaactaccccctg CNYPLAEHLTPTKHAIIQALgcagagcatctcacacccacaaagcatgcaattatccaggcctt VHLKNSQKASKACCVPTKLggtccacctcaagaattcccagaaagcttccaaagcctgctgtg EPISILYLDKGVVTYKFKYEtgcccacaaagctagagcccatctccatcctctatttagacaaag GMAVSECGCR (SEQ IDgcgtcgtcacctacaagtttaaatacgaaggcatggccgtctcc NO: 57) gaatgtggctgtaga(SEQ ID NO: 56) BMP-15(GDF-caagcagatggtatctcagctgaggttactgcctcttcctcaaaa QADGISAEVTASSSKHSGPE 9b)catagcgggcctgaaaataaccagtgttccctccaccctttccaa NNQCSLHPFQISFRQLGWDatcagcttccgccagctgggttgggatcactggatcattgctccc HWIIAPPFYTPNYCKGTCLRcctttctacaccccaaactactgtaaaggaacttgtctccgagtac VLRDGLNSPNHAIIQNLINQtacgcgatggtctcaattcccccaatcacgccattattcagaacct LVDQSVPRPSCVPYKYVPIStatcaatcagttggtggaccagagtgtcccccggccctcctgtgt VLMIEANGSILYKEYEGMIAcccgtataagtatgttccaattagtgtccttatgattgaggcaaatg ESCTCR (SEQ ID NO: 59)ggagtattttgtacaaggagtatgagggtat gattgctgagtcttgtacatgcaga (SEQ ID NO:58) GDF-1 gacgccgaacccgtgttgggcggcggccccgggggcgcttgt DAEPVLGGGPGGACRARRLcgcgcgcggcggctgtacgtgagcttccgcgaggtgggctgg YVSFREVGWHRWVIAPRGFcaccgctgggtcatcgcgccgcgcggcttcctggccaactact LANYCQGQCALPVALSGSGgccagggtcagtgcgcgctgcccgtcgcgctgtcggggtccg GPPALNHAVLRALMHAAAgggggccgccggcgctcaaccacgctgtgctgcgcgcgctca PGAADLPCCVPARLSPISVLtgcacgcggccgccccgggagccgccgacctgccctgctgc FFDNSDNVVLRQYEDMVVgtgcccgcgcgcctgtcgcccatctccgtgctcttctttgacaac DECGC (SEQ ID NO: 61)agcgacaacgtggtgctgcggcagtatgaggacatggtggtg gacgagtgcggctgccgc (SEQ IDNO: 60) GDF-3(Vgr-2) gcagccatccctgtccccaagctttcttgtaagaacctctgccacAAIPVPKLSCKNLCHRHQLF cgtcaccagctattcattaacttccgggacctgggttggcacaagINFRDLGWHKWIIAPKGFM tggatcattgcccccaaggggttcatggcaaattactgccatggANYCHGECPFSLTISLNSSN agagtgtcccttctcactgaccatctctctcaacagctccaattatYAFMQALMHAVDPEIPQA gctttcatgcaagccctgatgcatgccgttgacccagagatcccVCIPTKLSPISMLYQDNNDN ccaggctgtgtgtatccccaccaagctgtctcccatttccatgctcVILRHYEDMVVDECGCG taccaggacaataatgacaatgtcattctacgacattatgaagac (SEQ IDNO: 63) atggtagtcgatgaatgtgggtgtggg (SEQ ID NO: 62) GDF-5(BMP-gccccactggccactcgccagggcaagcgacccagcaagaa APLATRQGKRPSKNLKARC 14)ccttaaggctcgctgcagtcggaaggcactgcatgtcaacttca SRKALHVNFKDMGWDDWIaggacatgggctgggacgactggatcatcgcaccccttgagta IAPLEYEAFHCEGLCEFPLRcgaggctttccactgcgaggggctgtgcgagttcccattgcgct SHLEPTNHAVIQTLMNSMDcccacctggagcccacgaatcatgcagtcatccagaccctgat PESTPPTCCVPTRLSPISILFIgaactccatggaccccgagtccacaccacccacctgctgtgtg DSANNVVYKQYEDMVVEScccacgcggctgagtcccatcagcatcctcttcattgactctgcc CGCR (SEQ ID NO: 65)aacaacgtggtgtataagcagtatgaggacat ggtcgtggagtcgtgtggctgcagg (SEQ ID NO:64) GDF-6(BMP- acggccttcgccagtcgccatggcaagcggcacggcaagaagTAFASRHGKRHGKKSRLRC 13) tccaggctacgctgcagcaagaagcccctgcacgtgaacttcaSKKPLHVNFKELGWDDWII aggagctgggctgggacgactggattatcgcgcccctggagtaAPLEYEAYHCEGVCDFPLR cgaggcctatcactgcgagggtgtatgcgacttcccgctgcgctSHLEPTNHAIIQTLMNSMDP cgcacctggagcccaccaaccacgccatcatccagacgctgatGSTPPSCCVPTKLTPISILYID gaactccatggaccccggctccaccccgcccagctgctgcgtgAGNNVVYKQYEDMVVESC cccaccaaattgactcccatcagcattctatacatcgacgcggg GCR(SEQID NO: 67) caataatgtggtctacaagcagtacgaggacatggtggtggagt cgtgcggctgcagg(SEQ ID NO: 66) GDF-7(BMP- acggcgttggccgggacgcggacatcgcagggcagcggcgTALAGTRTSQGSGGGAGRG 12) ggggcgcgggccggggccacgggcgcaggggccggagccHGRRGRSRCSRKPLHVDFK gctgcagccgcaagccgttgcacgtggacttcaaggagctcggELGWDDWIIAPLDYEAYHC ctgggacgactggatcatcgcgccgctggactacgaggcgtacEGLCDFPLRSHLEPTNHAII cactgcgagggcctttgcgacttccctttgcgttcgcacctcgagQTLLNSMAPDAAPASCCVP cccaccaaccatgccatcattcagacgctgctcaactccatggcARLSPISILYIDAANNVVYK accagacgcggcgccggcctcctgctgtgtgccagcgcgcctQYEDMVVEACGCR (SEQ cagccccatcagcatcctctacatcgacgccgccaacaacgttg ID NO:69) tctacaagcaatacgaggacatggtggtggaggcctgcggctg cagg (SEQ ID NO: 68)GDF- gattttggtcttgactgtgatgagcactcaacagaatcacgatgct DFGLDCDEHSTESRCCRYP8(Myostatin) gtcgttaccctctaactgtggattttgaagcttttggatgggattggLTVDFEAFGWDWIIAPKRY attatcgctcctaaaagatataaggccaattactgctctggagagtKANYCSGECEFVFLQKYPH gtgaatttgtatttttacaaaaatatcctcatactcatctggtacaccTHLVHQANPRGSAGPCCTP aagcaaaccccagaggttcagcaggcccttgctgtactcccacTKMSPINMLYFNGKEQIIYG aaagatgtctccaattaatatgctatattttaatggcaaagaacaaKIPAMVVDRCGCS (SEQ ID ataatatatgggaaaattccagcgatggtagtagaccgctgtgg NO:71) gtgctca (SEQ ID NO: 70) GDF-9ggtcaggaaactgtcagttctgaattgaagaagcccttgggccc GQETVSSELKKPLGPASFNLagcttccttcaatctgagtgaatacttcagacaatttcttcttcccca SEYFRQFLLPQNECELHDFRaaatgagtgtgagctccatgactttagacttagctttagtcagctg LSFSQLKWDNWIVAPHRYNaagtgggacaactggattgtggctccgcacaggtacaaccctc PRYCKGDCPRAVGHRYGSPgatactgtaaaggggactgtccaagggcagttggacatcggtat VHTMVQNIIYEKLDSSVPRPggctctccagttcacaccatggtacagaacatcatctatgagaa SCVPAKYSPLSVLTIEPDGSIgctggactcctcagtgccaagaccgtcatgtgtacctgccaaat AYKEYEDMIATKCTCRacagccccttgagtgttttgaccattgagcccgatggctcaattg (SEQ ID NO: 73)cctataaagagtacgaagatatgatagctacaaagtgcacctgt cgt (SEQ ID NO: 72)GDF-10(BMP- aagacgatgcagaaagcccggaggaagcagtgggatgagcc KTMQKARRKQWDEPRVCS3b) gagggtgtgctcccggaggtacctgaaggtggacttcgcagac RRYLKVDFADIGWNEWIISPatcggctggaatgaatggataatctcaccgaaatcttttgatgcct KSFDAYYCAGACEFPMPKIactactgcgcgggagcatgtgagttccccatgcctaagatcgtt VRPSNHATIQSIVRAVGIIPGcgtccatccaaccatgccaccatccagagcattgtcagggctgt IPEPCCVPDKMNSLGVLFLDgggcatcatccctggcatcccagagccctgctgtgttcccgata ENRNVVLKVYPNMSVDTCagatgaactcccttggggtcctcttcctggatgagaat301cgg ACR (SEQ ID NO: 75)aatgtggttctgaaggtgtaccccaacatgtccgtggacacctgt gcctgccggtga (SEQ ID NO:74) GDF-11(BMP- aacctgggtctggactgcgacgagcactcaagcgagtcccgctNLGLDCDEHSSESRCCRYP 11) gctgccgatatcccctcacagtggactttgaggctttcggctgggLTVDFEAFGWDWIIAPKRY actggatcatcgcacctaagcgctacaaggccaactactgctccKANYCSGQCEYMFMQKYP ggccagtgcgagtacatgttcatgcaaaaatatccgcatacccaHTHLVQQANPRGSAGPCCT tttggtgcagcaggccaatccaagaggctctgctgggccctgttPTKMSPINMLYFNDKQQIIY gtacccccaccaagatgtccccaatcaacatgctctacttcaatgGKIPGMVVDRCGCS (SEQ acaagcagcagattatctacggcaagatccctggcatggtggtg ID NO:77) gatcgctgtggctgctct (SEQ ID NO: 76) GDF-15gcgcgcaacggggaccactgtccgctcgggcccgggcgttgc ARNGDDCPLGPGRCCRLHTtgccgtctgcacacggtccgcgcgtcgctggaagacctgggct VRASLEDLGWADWVLSPRgggccgattgggtgctgtcgccacgggaggtgcaagtgaccat EVQVTMCIGACPSQFRAANgtgcatcggcgcgtgcccgagccagttccgggcggcaaacat MHAQIKTSLHRLKPDTEPAgcacgcgcagatcaagacgagcctgcaccgcctgaagcccg PCCVPASYNPMVLIQKTDTacacggtgccagcgccctgctgcgtgcccgccagctacaatcc GVSLQTYDDLLAKDCHCIcatggtgctcattcaaaagaccgacaccggggtgtcgctccag (SEQ ID NO: 79)acctatgatgacttgttagccaaagactgccactgcata (SEQ ID NO: 78) Nodalcatcacttgccagacagaagtcaactgtgtcggaaggtcaagtt HHLPDRSQLCRKVKFQVDFccaggtggacttcaacctgatcggatggggctcctggatcatct NLIGWGSWIIYPKQYNAYRaccccaagcagtacaacgcctatcgctgtgagggcgagtgtcc CEGECPNPVGEEFHPTNHAtaatcctgttggggaggagtttcatccgaccaaccatgcatacat YIQSLLKRYQPHRVPSTCCAccagagtctgctgaaacgttaccagccccaccgagtcccttcca PVKTKPLSMLYVDNGRVLLcttgttgtgccccagtgaagaccaagccgctgagcatgctgtat DHHKDMIVEECGCL (SEQgtggataatggcagagtgctcctagatcaccataaagacatgat ID NO: 81)cgtggaagaatgtgggtgcctc (SEQ ID NO: 80) Activin-βAggcctggagtgcgacggcaaggtcaacatctgctgtaagaaac GLECDGKVNICCKKQFFVSagttctttgtcagtttcaaggacatcggctggaatgactggatcat FKDIGWNDWIIAPSGYHANtgctccctctggctatcatgccaactactgcgagggtgagtgcc YCEGECPSHIAGTSGSSLSFcgagccatatagcaggcacgtccgggtcctcactgtccttccac HSTVINHYRMRGHSPFANLtcaacagtcatcaaccactacgcatgcggccatagcccctttgc KSCCVPTKLRPMSMLYYDDcaacctcaaatcgtgctgtgtgcccaccaagctgagacccatgt GQNIIKKDIQNMIVEECGCSccatgttgtactatgatgatggtcaaaacatcatcaaaaaggaca (SEQ ID NO: 83)ttcagaacatgatcgtggaggagtgcgggtgctcc (SEQ ID NO: 82) Activin-βBggcctggagtgcgatggccggaccaacctctgttgcaggcaac GLECDGRTNLCCRQQFFIDFagttcttcattgacttccgcctcatcggctggaacgactggatcat RLIGWNDWIIAPTGYYGNYagcacccaccggctactacggcaactactgtgagggcagctgc CEGSCPAYLAGVPGSASSFccagcctacctggcaggggtccccggctctgcctcctccttcca HTAVVNQYRMRGLNPGTVcacggctgtggtgaaccagtaccgcatgcggggtctgaacccc NSCCIPTKLSTMSMLYFDDEggcacggtgaactcctgctgcattcccaccaagctgagcaccat YNIVKRDVPNMIVEECGCAgtccatgctgtacttcgatgatgagtacaacatcgtcaagcggg (SEQ ID NO: 85)acgtgcccaacatgattgtggaggagtgcggctgcgcc (SEQ ID NO: 84) Activin-βCggcatcgactgccaaggagggtccaggatgtgctgtcgacaa GIDCQGGSRMCCRQEFFVDgagttttttgtggacttccgtgagattggctggcacgactggatca FREIGWHDWIIQPEGYAMNtccagcctgagggctacgccatgaacttctgcatagggcagtgc FCIGQCPLHIAGMPGIAASFccactacacatagcaggcatgcctggtattgctgcctcctttcac HTAVLNLLKANTAAGTTGactgcagtgctcaatcttctcaaggccaacacagctgcaggcac GGSCCVPTARRPLSLLYYDcactggagggggctcatgctgtgtacccacggcccggcgccc RDSNIVKTDIPDMVVEACGcctgtctctgctctattatgacagggacagcaacattgtcaagact CS (SEQ ID NO: 87)gacatacctgacatggtagtagaggcctgtgggtgcagt (SEQ ID NO: 86) Activin-βEacccccacctgtgagcctgcgacccccttatgttgcaggcgag TPTCEPATPLCCRRDHYVDaccattacgtagacttccaggaactgggatggcgggactggat FQELGWRDWILQPEGYQLNactgcagcccgaggggtaccagctgaattactgcagtgggcag YCSGQCPPHLAGSPGIAASFtgccctccccacctggctggcagcccaggcattgctgcctctttc HSAVFSLLKANNPWPASTScattctgccgtcttcagcctcctcaaagccaacaatccttggcct CCVPTARRPLSLLYLDHNGgccagtacctcctgttgtgtccctactgcccgaaggcccctctct NVVKTDVPDMVVEACGCSctcctctacctggatcataatggcaatgtggtcaagacggatgtg (SEQ ID NO: 89)ccagatatggtggtggaggcctgtggctgcagc (SEQ ID NO: 88) Inhibin-αtcaactcccctgatgtcctggccttggtctccctctgctctgcgcc STPLMSWPWSPSALRLLQRtgctgcagaggcctccggaggaaccggctgcccatgccaact PPEEPAAHANCHRVALNISFgccacagagtagcactgaacatctccttccaggagctgggctg QELGWERWIVYPPSFIFHYCggaacggtggatcgtgtaccctcccagtttcatcttccactactgt HGGCGLHIPPNLSLPVPGAPcatggtggttgtgggctgcacatcccaccaaacctgtcccttcca PTPAQPYSLLPGAQPCCAALgtccctggggctccccctaccccagcccagccctactccttgct PGTMRPLHVRTTSDGGYSFgccaggggcccagccctgctgtgctgctctcccagggaccatg KYETVPNLLTQHCACI (SEQaggcccctacatgtccgcaccacctcggatggaggttactctttc ID NO: 91)aagtatgagacagtgcccaaccttctcacgcagcactgtgcttgt atc (SEQ ID NO: 90) TGF-β1gccctggacaccaactattgcttcagctccacggagaagaactg ALDTNYCFSSTEKNCCVRQctgcgtgcggcagctgtacattgacttccgcaaggacctcggct LYIDFRKDLGWKWIHEPKGggaagtggatccacgagcccaagggctaccatgccaacttctg YHANFCLGPCPYIWSLDTQcctcgggccctgcccctacatttggagcctggacacgcagtac YSKVLALYNQHNPGASAAPagcaaggtcctggccctgtacaaccagcataacccgggcgcct CCVPQALEPLPIVYYVGRKPcggcggcgccgtgctgcgtgccgcaggcgctggagccgctg KVEQLSNMIVRSCKCS (SEQcccatcgtgtactacgtgggccgcaagcccaaggtggagcag ID NO: 93)ctgtccaacatgatcgtgcgctcctgcaagtgcagc (SEQ ID NO: 92 TGF-β2gctttggatgcggcctattgctttagaaatgtgcaggataattgct ALDAAYCFRNVQDNCCLRPgcctacgtccactttacattgatttcaagagggatctagggtgga LYIDFKRDLGWKWIHEPKGaatggatacacgaacccaaagggtacaatgccaacttctgtgct YNANFCAGACPYLWSSDTggagcatgcccgtatttatggagttcagacactcagcacagcag QHSRVLSLYNTINPEASASPggtcctgagcttatataataccataaatccagaagcatctgcttct CCVSQDLEPLTILYYIGKTPccttgctgcgtgtcccaagatttagaacctctaaccattctctacta KIEQLSNMIVKSCKCS (SEQcattggcaaaacacccaagattgaacagctttctaatatgattgta ID NO: 95)aagtcttgcaaatgcagc (SEQ ID NO: 94) TGF-β3gctttggacaccaattactgcttccgcaacttggaggagaactgc ALDTNYCFRNLEENCCVRPtgtgtgcgccccctctacattgacttccgacaggatctgggctgg LYIDFRQDLGWKWVHEPKaagtgggtccatgaacctaagggctactatgccaacttctgctca GYYANFCSGPCPYLRSADTggcccttgcccatacctccgcagtgcagacacaacccacagca THSTVLGLYNTLNPEASASPcggtgctgggactgtacaacactctgaaccctgaagcatctgcc CCVPQDLEPLTILYYVGRTPtcgccttgctgcgtgccccaggacctggagcccctgaccatcct KVEQLSNMVVKSCKCSgtactatgttgggaggacccccaaagtggagcagctctccaac (SEQ ID NO: 97)atggtggtgaagtcttgtaaatgtagc (SEQ ID NO: 96)

1. A recombinant polypeptide comprising: at least two peptide segments,a first segment of the polypeptide comprising a sequence having at least80% identity to a first TGF-beta family protein and a second segmentcomprising a sequence having at least 80% identity to a second TGF-betafamily protein or a combination thereof, wherein the segments areoperably linked and have activity of at least one of the first or secondparental TGF-beta family protein, or activity of new in vivo signalingand cellular property.
 2. The polypeptide of claim 1, wherein thepolypeptide comprises 5 or 6 domains, having the general sequence orderof 1-2-3-4-5-6, wherein each domain can be recombined with at least oneother domain from at least one other TGF-beta family polypeptide in asequential order.
 3. The polypeptide of claim 2, wherein the polypeptidecomprises 5 domains.
 4. The polypeptide of claim 2, wherein domain 3 isonly shuffled when at least one other domain is shuffled in a chimericpolypeptide.
 5. The polypeptide of claim 2, wherein the domains are asset forth in Table A.
 6. The polypeptide of claim 1, wherein thepolypeptide comprises an N-terminal segment from BMP-2.
 7. Thepolypeptide of claim 1, wherein the at least two polypeptide segmentscomprise 6 peptide segments operably linked N- to C-terminal.
 8. Thepolypeptide of claim 7, wherein each of the first and second TGF-betafamily proteins have structural similarity and wherein each segmentcorresponds to a structural motif of the segment.
 9. The polypeptide ofclaim 1, wherein the first TGF-beta family protein is BMP-2 and thesecond TGF-beta family protein is activin.
 10. The polypeptide of claim7, wherein the first TGF-beta family protein is BMP-2 and the secondTGF-beta family protein is activin or other family member.
 11. Thepolypeptide of claim 10, wherein the segments of the BMP-2 proteincomprise segment 1: amino acid residue from about 1 to about x₁ of SEQID NO:2 (“1b”); segment 2 is from about amino acid residue x₁ to aboutx₂ of SEQ ID NO:2 (“2b”); segment 3 is from about amino acid residue x₂to about x₃ of SEQ ID NO:2 (“3b”); segment 4 is from about amino acidresidue x₃ to about x₄ of SEQ ID NO:2 (“4b”); segment 5 is from aboutamino acid residue x₄ to about x₅ of SEQ ID NO:2 (“5b”); and segment 6is from about amino acid residue x₅ to about x₆ of SEQ ID NO:2 (“6b”);and wherein: x₁ is residue 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35of SEQ ID NO:2; x₂ is residue 45, 46, 47, or 48 of SEQ ID NO:2; x₃ isresidue 65, 66, 67, or 68 of SEQ ID NO:2; x₄ is residue 76, 77, 78, 79,80, 81 or 82 of SEQ ID NO:2; x₅ is residue 88, 89, 90, 91, 92, 93, or 94of SEQ ID NO:2; and x₆ is residue 112, 113, or 114 or SEQ ID NO:2,corresponding to the C-terminus of BMP-2; and wherein the segments ofthe activin protein comprise segment 1, amino acid residue from about 1to about x₁ of SEQ ID NO:5 (“1a”); segment 2 is from about amino acidresidue x₁ to about x₂ of SEQ ID NO:5 (“2a”); segment 3 is from aboutamino acid residue x₂ to about x₃ of SEQ ID NO:5 (“3a”); segment 4 isfrom about amino acid residue x₃ to about x₄ of SEQ ID NO:5 (“4a”);segment 5 is from about amino acid residue x₄ to about x₅ of SEQ ID NO:5(“5a”); and segment 6 is from about amino acid residue x₅ to about x₆ ofSEQ ID NO:5 (“6a”); and wherein: x₁ is residue 22, 23, 24, 25, 26, 27,28, 29, 30, 31 or 32 of SEQ ID NO:5; x₂ is residue 42, 43, 44, or 45 ofSEQ ID NO:5; x₃ is residue 61, 62, 63, or 64 of SEQ ID NO:5; x₄ isresidue 78, 79, 80, 81, 82, 83 or 84 of SEQ ID NO:5; x₅ is residue 90,91, 92, 93, 94, 95 or 96 of SEQ ID NO:5; and x₆ is residue 114, 115, or116 or SEQ ID NO:5; and wherein the polypeptide has an order of segment1-segment 2-segment 3-segment 4-segment 5-segment
 6. 12. The polypeptideof claim 10, wherein the polypeptide comprises a sequence of segmentsselected from the group consisting of 1b2b3b4b5b6a; 1b2b3b4b5a6a;1b2b3b4b5a6b; 1b2b3a4a5a6a; 1b2b3a4a5b6a; 1b2a3a4a5a6a; 1b2a3a4a5a6aL66V/V67I; 1b(1a_II)2a3a4a5a6a; 1b2a3a4a5a6b; 1b2a3a4a5b6b;1b2a3a4a5b6a; 1b2a3b4b5b6a; 1b2a3b4b5a6a; and 1b2a3b4b5a6b.
 13. Thepolypeptide of claim 1, wherein the polypeptide comprises 80%, 90%, 95%,98% or 99% identity to a sequence as set forth in SEQ ID NO:7, 9, 11,13, 15, 17, 19, 1, 23, 25, 2, 29, 31, 33, 35, 37, 39 or 41 and whereinthe polypeptide modulates the SMAD or DAXX pathway.
 14. The polypeptideof claim 13, wherein the polypeptide comprises a sequence selected fromthe group consisting of SEQ ID NO:7, 9, 11, 13, 15, 17, 19, 1, 23, 25,2, 29, 31, 33, 35, 37, 39 and
 41. 15. A chimeric TGF-beta familypolypeptide comprising a segment of a first TGF-beta family proteinoperably linked to a segment of a second different TGF-beta familyprotein to provide a chimeric polypeptide having SMAD or DAXX modulatingactivity.
 16. The polypeptide of claim 15, wherein the first TGF-betafamily protein is BMP-2 and the second TGF-beta family protein isactivin.
 17. The polypeptide of claim 16, wherein the segments of theBMP-2 protein comprise segment 1: amino acid residue from about 1 toabout x₁ of SEQ ID NO:2 (“1b”); segment 2 is from about amino acidresidue x₁ to about x₂ of SEQ ID NO:2 (“2b”); segment 3 is from aboutamino acid residue x₂ to about x₃ of SEQ ID NO:2 (“3b”); segment 4 isfrom about amino acid residue x₃ to about x₄ of SEQ ID NO:2 (“4b”);segment 5 is from about amino acid residue x₄ to about x₅ of SEQ ID NO:2(“5b”); and segment 6 is from about amino acid residue x₅ to about x₆ ofSEQ ID NO:2 (“6b”); and wherein: x₁ is residue 25, 26, 27, 28, 29, 30,31, 32, 33, 34, or 35 of SEQ ID NO:2; x₂ is residue 45, 46, 47, or 48 ofSEQ ID NO:2; x₃ is residue 65, 66, 67, or 68 of SEQ ID NO:2; x₄ isresidue 76, 77, 78, 79, 80, 81 or 82 of SEQ ID NO:2; x₅ is residue 88,89, 90, 91, 92, 93, or 94 of SEQ ID NO:2; and x₆ is residue 112, 113, or114 or SEQ ID NO:2, corresponding to the C-terminus of BMP-2; andwherein the segments of the activin protein comprise segment 1, aminoacid residue from about 1 to about x₁ of SEQ ID NO:5 (“1a”); segment 2is from about amino acid residue x₁ to about x₂ of SEQ ID NO:5 (“2a”);segment 3 is from about amino acid residue x₂ to about x₃ of SEQ ID NO:5(“3a”); segment 4 is from about amino acid residue x₃ to about x₄ of SEQID NO:5 (“4a”); segment 5 is from about amino acid residue x₄ to aboutx₅ of SEQ ID NO:5 (“5a”); and segment 6 is from about amino acid residuex₅ to about x₆ of SEQ ID NO:5 (“6a”); and wherein: x₁ is residue 22, 23,24, 25, 26, 27, 28, 29, 30, 31 or 32 of SEQ ID NO:5; x₂ is residue 42,43, 44, or 45 of SEQ ID NO:5; x₃ is residue 61, 62, 63, or 64 of SEQ IDNO:5; x₄ is residue 78, 79, 80, 81, 82, 83 or 84 of SEQ ID NO:5; x₅ isresidue 90, 91, 92, 93, 94, 95 or 96 of SEQ ID NO:5; and x₆ is residue114, 115, or 116 or SEQ ID NO:5; and wherein the polypeptide has anorder of segment 1-segment 2-segment 3-segment 4-segment 5-segment 6.18. The polypeptide of claim 16, wherein the polypeptide comprises asequence of segments selected from the group consisting of 1b2b3b4b5b6a;1b2b3b4b5a6a; 1b2b3b4b5a6b; 1b2b3a4a5a6a; 1b2b3a4a5b6a; 1b2a3a4a5a6a;1b2a3a4a5a6a L66V/V67I; 1b(1a_II)2a3a4a5a6a; 1b2a3a4a5a6b; 1b2a3a4a5b6b;1b2a3a4a5b6a; 1b2a3b4b5b6a; 1b2a3b4b5a6a; and 1b2a3b4b5a6b.
 19. Apolynucleotide encoding a polypeptide of claim 1 or
 15. 20. Thepolynucleotide of claim 19, wherein the polynucleotide comprisessequences from a plurality of TGF-beta family polynucleotides operablylinked to encode a functional chimeric polypeptide having SMAD or DAXXmodulating activity.
 21. A polynucleotide encoding a polypeptide ofclaim
 20. 22. A polynucleotide comprising a sequence selected from thegroup consisting of SEQ ID NO:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 24, 26, 28, and
 40. 23. A vector comprising a polynucleotideof claim
 21. 24. A host cell comprising the vector of claim
 23. 25. Ahost cell comprising a polynucleotide of claim
 21. 26. A method ofproducing a chimeric TGF-beta polypeptide comprising (a) aligning thesequences of at least two TGF-beta family member proteins; (b)identifying structurally related domains of the at least two familymember proteins; (c) identifying points of cross-over of the at leasttwo TGF-beta proteins comprising regions at either or both ends of thestructurally related domains and wherein the regions comprise at least80%, 95%, 98%, 99% or 100% sequence identity over at least 5 consecutiveamino acids; (d) generating a chimeric TGF-beta polypeptide comprisingat least one domain from a first TGF-beta family member protein and atleast one domain from a second TGF-beta family member protein whereinthe domains are linked at the points of cross-over; and (e) assaying thechimeric TGF-beta polypeptide for type 1 and type II ligand bindingcapacity.
 27. A chimeric polypeptide produced from the method of claim26.
 28. A method of modulating cell proliferation or activity associatedwith the Smad or DAXX pathway comprising contacting a cell with achimeric polypeptide of claim
 1. 29. A method of treating a disease ordisorder associated with bone, cartilage, neurological tissue, cardiactissue, skeletal muscle or endocrine tissue comprising contacting thetissue with a chimeric polypeptide of claim
 1. 30. A method of treatinga cell proliferative disease or disorder comprising contacting a cellhaving the cell proliferative disease or disorder with a chimericpolypeptide of claim 1.